Aflibercept formulations and uses thereof

ABSTRACT

Ophthalmic formulations comprising aflibercept are disclosed that are suitable for a method of treatment of an eye disorder or disease by intravitreal or topical administration.

This is a U.S. national phase application under 35 U.S.C. § 371 ofUnited States Patent Cooperation Treaty Application No.PCT/US2017/062521, filed Nov. 20, 2017, which claims priority from U.S.Provisional Patent Application Ser. No. 62/497,584, filed in the UnitedStates Patent and Trademark Office on Nov. 21, 2016, and whichincorporates by reference those PCT and Provisional applications intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Nov. 10, 2017, is namedJUST0271_SL.txt and is 4,093 bytes in size.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to pharmaceutical formulations of afliberceptfusion protein suitable for ophthalmic administration.

2. Discussion of the Related Art

Aflibercept is a recombinant fusion protein that includes two maincomponents: the vascular endothelial growth factor (VEGF) bindingportions from the extracellular domains of human VEGF receptors 1 and 2,fused to the Fc portion of human IgG1. (See, Papadopoulos et al.,Modified chimeric polypeptides with improved pharmacokinetic properties,WO 00/75319 A1; U.S. Pat. No. 7,070,959B2). Structurally, aflibercept isa dimeric glycoprotein with a protein molecular weight of about 96.9kilo Daltons (kDa). It contains approximately 15% glycosylation to givea total molecular weight of approximately 115 kDa. All five putativeN-glycosylation sites on each polypeptide chain predicted by the primarysequence can be occupied with carbohydrate and exhibit some degree ofchain heterogeneity, including heterogeneity in terminal sialic acidresidues.

The United States Food and Drug Administration (FDA) approvedaflibercept for marketing in November 2011, and the European MedicinesAgency (EMA) approved in November 2012.

Aflibercept, under the brand name Eylea® (Regeneron Pharmaceuticals,Inc.) is used as an ophthalmic agent in the treatment of eye disordersor diseases, e.g., macular edema following Central Retinal VeinOcclusion (CRVO), Central Retinal Vein Occlusion (CRVO), Branch RetinalVein Occlusion (BRVO), Neovascular (Wet) Age-Related MacularDegeneration (AMD), Impaired vision due to Myopic ChoroidalNeovascularisation, Diabetic Macular Edema (DME), Diabetic Retinopathy(DR) in patients with DME, and neovascular Age-Related MacularDegeneration (AMD).

Ziv-aflibercept, under the brand name Zaltrap® (RegeneronPharmaceuticals, Inc.), was developed as an injection for treatment ofmetastatic colorectal cancer.

Known formulations for aflibercept include those described by Furfine etal. (Furfine et al., VEGF antagonist formulations for intravitrealadministration, U.S. Pat. Nos. 8,092,803; 9,580,489; EP 2364691B1;WO2007149334A2) and by Dix et al. (Dix et al., VEGF AntagonistFormulations, WO2006104852 A2; U.S. Pat. Nos. 8,921,316; 9,636,400).

There is still a need for formulations of aflibercept with enhancedstability, which the present invention provides.

SUMMARY OF THE INVENTION

The present invention relates to an ophthalmic formulation ofaflibercept, which formulation includes: (a) aflibercept in aconcentration of 5-100 mg/mL; (b) a buffer at 5-50 mM concentration; (c)a non-ionic surfactant; (d) a tonicifying agent selected from the groupconsisting of a polyol and an amino acid, or in some embodiments both apolyol and an amino acid, with the formulation having a final osmolalityof about 300 mOsm/kg (i.e., 300±50 mOsm/kg). The concentration ofchloride anion (CO in the inventive ophthalmic formulation is less thanabout 10 mM, and in some embodiments less than about 5 mM or less thanabout 1 mM; and the pH of the formulation is about pH 5.0 to about pH6.5. The inventive ophthalmic formulation is suitable for intravitrealor topical administration. The inventive aflibercept-containingophthalmic formulations have stability characteristics, e.g.,significantly reduced aggregation over time, and visual characteristicsas favorable, or more favorable, than other known ophthalmicformulations of aflibercept, for example, formulations containing addedsodium chloride. Formulations of the invention can also be lyophilizedand reconstituted, if desired.

The ophthalmic formulations of the present invention can be used asmedicinal ophthalmic agents in a method of treatment of an eye disorderor disease, e.g., macular edema following Retinal Vein Occlusion (RVO),Central Retinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion(BRVO), Neovascular (Wet) Age-Related Macular Degeneration (AMD),Impaired vision due to Myopic Choroidal Neovascularisation, DiabeticMacular Edema (DME), Diabetic Retinopathy (DR) in patients with DME, andneovascular Age-Related Macular Degeneration (AMD). Administration ofthe inventive ophthalmic formulation can be by intravitreal injectionor, in some cases, by topical administration to the eye, as medicallyappropriate. The inventive formulations can be used for the treatment ofthese eye disorders or diseases and used in the preparation ofmedicaments for treatment of these eye disorders and diseases.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description of Embodiments. The entire document isintended to be related as a unified disclosure, and it should beunderstood that all combinations of features described herein arecontemplated, even if the combination of features are not found togetherin the same sentence, or paragraph, or section of this document.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations defined by specific paragraphs above. For example,certain aspects of the invention that are described as a genus, and itshould be understood that every member of a genus is, individually, anaspect of the invention. Also, aspects described as a genus or selectinga member of a genus, should be understood to embrace combinations of twoor more members of the genus. Although the applicant(s) invented thefull scope of the invention described herein, the applicants do notintend to claim subject matter described in the prior art work ofothers. Therefore, in the event that statutory prior art within thescope of a claim is brought to the attention of the applicants by aPatent Office or other entity or individual, the applicant(s) reservethe right to exercise amendment rights under applicable patent laws toredefine the subject matter of such a claim to specifically exclude suchstatutory prior art or obvious variations of statutory prior art fromthe scope of such a claim. Variations of the invention defined by suchamended claims also are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results from subvisible particle analysis, conducted bysmall volume HIAC analysis. The 10-μm particle results showed that allsamples had low levels of particles except Formulation 7, a formulationthat exhibited increasing levels of particles during storage.

FIG. 2 shows results from subvisible particle analysis, conducted bysmall volume HIAC analysis. The 25-μm results indicated that all sampleshad low or undetectable levels of particles. The error bars,representing the standard deviation of three replicate measurements, arelarger than the number of particles observed.

FIG. 3 shows results from size exclusion high performance liquidchromatography of samples stored at 4° C. Apart from Formulation 8, allformulations showed similar rates of HMW formation.

FIG. 4 shows results from size exclusion high performance liquidchromatography of samples stored at 30° C. Apart from Formulation 8, allformulations showed similar rates of HMW formation.

FIG. 5 shows results of the reduced CE-SDS analysis for the 4° C.storage condition. All formulations exhibited similar levels of percentpurity during the 7 weeks of testing.

FIG. 6 shows results of the reduced CE-SDS analysis for the 30° C.storage condition. All formulations, except for Formulation 7, exhibitedsimilar levels of percent purity during 30° C. storage. Formulation 7exhibited a consistent decrease in percent purity over time.

FIG. 7 shows results of the cIEF analysis, used to assess theaflibercept charge distribution during 4° C. storage. All formulationsexhibited similar levels of percent basic species over time.

FIG. 8 shows results of cIEF analysis, used to assess the afliberceptcharge distribution during 4° C. storage. All formulations exhibitedsimilar levels of percent acidic species over time.

FIG. 9 shows results of cIEF analysis, used to assess the afliberceptcharge distribution during 30° C. storage. All formulations exhibitedsimilar levels of percent basic species over time.

FIG. 10 shows results of cIEF analysis, used to assess the afliberceptcharge distribution during 30° C. storage. All formulations exhibitedsimilar levels of percent acidic species over time.

FIG. 11 shows HMW formation in various aflibercept formulations storedat 30° C., as measured by SE-HPLC. (See, Table 4 for formulationabbreviations.)

FIG. 12 shows results of a stability comparison of recombinantlyproduced aflibercept at 30° C. compared to commercially obtained Eylea®(aflibercept; Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.). Therecombinanly produced aflibercept was made by Just Biotherapeutics(Seattle, Wash.) and formulated in 10 mM acetate, 3% (w/v) proline, pH5.2, 0.1% (w/v) poloxamer formulation (A52ProP1-0.1).

FIG. 13 illustrates the effect of 100 mM sodium chloride (“salt”),compared to a control (minus any added sodium chloride), on thestability of recombinantly produced aflibercept in 10 mM acetate, 3%(w/v) proline, pH 5.2 formulation (A52ProP1-0.1), during storage at 30°C.

DETAILED DESCRIPTION OF EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Thus, as usedin this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlyindicates otherwise. For example, reference to “a protein” includes aplurality of proteins; reference to “a cell” includes populations of aplurality of cells.

The present invention relates to an aqueous ophthalmic formulation,suitable for intravitreal or topical administration to a patient, whichformulation includes aflibercept, which is also known commercially asEylea®. Aflibercept is an assembly of two identical fusion polypeptidechains having the aflibercept amino acid sequence (SEQ ID NO:1),typically produced most conveniently by recombinant DNA expressiontechnology. The aflibercept amino acid sequence is the following:

SEQ ID NO: 1 SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP

ITVTLKKFPLDTL IPDGKRIIWDSRKGFIIS

ATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTIIDVVLSPSHGIELSVGEKLVL

CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK

STFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG//

Disulfide bridges are expected between the cysteine residues atfollowing amino acid positions of SEQ ID NO:1 (underlined cysteine (C)residues shown in SEQ ID NO:1, above):

30-79 (intrachain)

124-185 (intrachain)

211-211 (interchain)

214-214 (interchain)

246-306 (intrachain)

352-410 (intrachain).

The two fusion polypeptide chains of aflibercept are covalently linkedby disulfide linkage at amino acid positions 211 and 214 of SEQ IDNO: 1. The fusion protein is typically glycosylated, with N-glycancovalently linked at asparagine residues at positions 36, 68, 123, 196,and 282 of SEQ ID NO:1 (bold/italicized asparagine (N) residues shown inSEQ ID NO:1 above). “Aflibercept” within the scope of the invention alsoincludes embodiments in which one, both, or none, of the fusionpolypeptide chains has the amino acid sequence SEQ ID NO:1 with anadditional carboxy-terminal lysine (K) residue. The concentration ofaflibercept in the inventive ophthalmic formulation is about 20 mg/mL toabout 80 mg/mL, or about 30 mg/mL to about 50 mg/mL; for example, aconcentration of about 40 mg/mL is useful in many embodiments of theformulation.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon processing (e.g., ultrafiltration,diafiltration, other filtering steps, vial filling), transportation,and/or storage of the drug substance and/or drug product containingaflibercept. Together, the physical, chemical and biological stabilityof the protein in a formulation embody the “stability” of the proteinformulation, e.g., the aflibercept formulation, which is specific to theconditions under which the formulated drug product (DP) is stored. Forinstance, a drug product stored at subzero temperatures would beexpected to have no significant change in either chemical, physical orbiological activity while a drug product stored at 40° C. would beexpected to have changes in its physical, chemical and biologicalactivity with the degree of change dependent on the time of storage forthe drug substance or drug product. The configuration of the proteinformulation can also influence the rate of change. For instance,aggregate formation is highly influenced by protein concentration withhigher rates of aggregation observed with higher protein concentration.Excipients are also known to affect stability of the drug product with,for example, addition of salt increasing the rate of aggregation forsome proteins while other excipients such as sucrose are known todecrease the rate of aggregation during storage. Instability is alsogreatly influenced by pH giving rise to both higher and lower rates ofdegradation depending on the type of modification and pH dependence.

Various analytical techniques for measuring protein stability areavailable in the art and are reviewed, e.g., in Wang, W. (1999),Instability, stabilization and formulation of liquid proteinpharmaceuticals, Int J Pharm 185:129-188. Stability can be measured at aselected temperature for a selected time period. For rapid screening,for example, the formulation may be kept at 40° C. for 2 weeks to 1month, at which time stability is measured. Where the formulation is tobe stored at 2-8° C., generally the formulation should be stable at 30°C. for at least 1 month, or 40° C. for at least a week, and/or stable at2-8° C. for at least two years.

A protein “retains its physical stability” in a pharmaceuticalformulation if it shows minimal signs of changes to the secondary and/ortertiary structure (i.e., intrinsic structure), or aggregation, and/orprecipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion high performance liquid chromatography, or other suitablemethods. Physical instability of a protein, i.e., loss of physicalstability, can be caused by oligomerization resulting in dimer andhigher order aggregates, subvisible, and visible particle formation, andprecipitation. The degree of physical degradation can be ascertainedusing varying techniques depending on the type of degradant of interest.Dimers and higher order soluble aggregates can be quantified using sizeexclusion chromatography, while subvisible particles may be quantifiedusing light scattering, light obscuration or other suitable techniques.In one embodiment, the stability of the protein is determined accordingto the percentage of aflibercept monomer protein in the solution, with alow percentage of degraded (e.g., fragmented) and/or aggregated protein.An “aflibercept monomer” means an assembly of two polypeptide chainshaving the aflibercept amino acid sequence (SEQ ID NO:1), with orwithout an additional carboxy-terminal lysine residue on any of thepolypeptide chains. In an “aflibercept monomer,” the two afliberceptpolypeptide chains are assembled through association and disulfidecrosslinks of the immunoglobulin Fc domain portions of the sequences, asnoted hereinabove. For example, an aqueous formulation comprising astable protein may include (as a percentage of total protein) at least95% aflibercept monomer, at least 96% aflibercept monomer, at least 97%aflibercept monomer, at least 98% aflibercept monomer, or at least 99%aflibercept monomer protein. Alternatively, an aqueous formulation ofthe invention may include (as a percentage of total protein) about 5%aggregate and/or degraded aflibercept protein.

A protein “retains its chemical stability” in a pharmaceuticalformulation, if the chemical stability at a given time is such thatcovalent bonds are not made or broken, resulting in changes to theprimary structure of the protein component, e.g., aflibercept. Changesto the primary structure may result in modifications of the secondaryand/or tertiary and/or quarternary structure of the protein and mayresult in formation of aggregates or reversal of aggregates alreadyformed. Typical chemical modifications can include isomerization,deamidation, N-terminal cyclization, backbone hydrolysis, methionineoxidation, tryptophan oxidation, histidine oxidation, beta-elimination,disulfide formation, disulfide scrambling, disulfide cleavage, and otherchanges resulting in changes to the primary structure including D-aminoacid formation. Chemical instability, i.e., loss of chemical stability,may be interrogated by a variety of techniques including ion-exchangechromatography, capillary isoelectric focusing, analysis of peptidedigests and multiple types of mass spectrometric techniques. Chemicalstability can be assessed by detecting and quantifying chemicallyaltered forms of the protein. Chemical alteration may involve sizemodification (e.g. clipping) which can be evaluated using size exclusionchromatography, SDS-PAGE and/or matrix-assisted laser desorptionionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example.Other types of chemical alteration include charge alteration (e.g.occurring as a result of deamidation) which can be evaluated bycharge-based methods, such as, but not limited to, ion-exchangechromatography, capillary isoelectric focusing, or peptide mapping.

Loss of physical and/or chemical stability may result in changes tobiological activity as either an increase or decrease of a biologicalactivity of interest, depending on the modification and the proteinbeing modified. A protein “retains its biological activity” in apharmaceutical formulation, if the biological activity of the protein ata given time is within about 30% of the biological activity exhibited atthe time the pharmaceutical formulation was prepared. Activity isconsidered decreased if the activity is less than 70% of its startingvalue. Biological assays may include both in vivo and in vitro basedassays such as ligand binding, potency, cell proliferation or othersurrogate measure of its biopharmaceutical activity. As an example,biological activity of aflibercept can be estimated using an in vitroligand binding assay such as inhibition of anti-placental growth factorbinging to PGF by ELISA or human umbilical vein endothelial cell (HUVEC)proliferation assay.

Aflibercept for use in the invention is typically produced byrecombinant expression technology. The term “recombinant” indicates thatthe material (e.g., a nucleic acid or a polypeptide) has beenartificially or synthetically (i.e., non-naturally) altered by humanintervention. The alteration can be performed on the material within, orremoved from, its natural environment or state. For example, a“recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, DNA shuffling or other well known molecularbiological procedures. Examples of such molecular biological proceduresare found in Maniatis et al., Molecular Cloning. A Laboratory Manual.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). A“recombinant DNA molecule,” is comprised of segments of DNA joinedtogether by means of such molecular biological techniques. The term“recombinant protein” or “recombinant polypeptide” as used herein refersto a protein molecule which is expressed using a recombinant DNAmolecule. A “recombinant host cell” is a cell that contains and/orexpresses a recombinant nucleic acid. Recombinant DNA molecules usefulin expressing aflibercept fusion protein are described, e.g., byPapadopoulos et al., Modified Chimeric Polypeptides with ImprovedPharmacokinetic Properties, U.S. Pat. No. 7,070,959 B2; and WO 00/75319A1).

The term “naturally occurring,” where it occurs in the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

The term “control sequence” or “control signal” refers to apolynucleotide sequence that can, in a particular host cell, affect theexpression and processing of coding sequences to which it is ligated.The nature of such control sequences may depend upon the host organism.In particular embodiments, control sequences for prokaryotes may includea promoter, a ribosomal binding site, and a transcription terminationsequence. Control sequences for eukaryotes may include promoterscomprising one or a plurality of recognition sites for transcriptionfactors, transcription enhancer sequences or elements, polyadenylationsites, and transcription termination sequences. Control sequences caninclude leader sequences and/or fusion partner sequences. Promoters andenhancers consist of short arrays of DNA that interact specifically withcellular proteins involved in transcription (Maniatis, et al., Science236:1237 (1987)). Promoter and enhancer elements have been isolated froma variety of eukaryotic sources including genes in yeast, insect andmammalian cells and viruses (analogous control elements, i.e.,promoters, are also found in prokaryotes). The selection of a particularpromoter and enhancer depends on what cell type is to be used to expressthe protein of interest. Some eukaryotic promoters and enhancers have abroad host range while others are functional in a limited subset of celltypes (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986)and Maniatis, et al., Science 236:1237 (1987)).

A “promoter” is a region of DNA including a site at which RNA polymerasebinds to initiate transcription of messenger RNA by one or moredownstream structural genes. Promoters are located near thetranscription start sites of genes, on the same strand and upstream onthe DNA (towards the 5′ region of the sense strand). Promoters aretypically about 100-1000 bp in length.

An “enhancer” is a short (50-1500 bp) region of DNA that can be boundwith one or more activator proteins (transcription factors) to activatetranscription of a gene.

The terms “in operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced. Forexample, a control sequence in a vector that is “operably linked” to aprotein coding sequence is ligated thereto so that expression of theprotein coding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of two or more amino acids linked covalently throughpeptide bonds. The terms do not refer to a specific length of theproduct. Thus, “peptides,” and “oligopeptides,” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. In addition, proteinfragments, analogs, mutated or variant proteins, fusion proteins and thelike are included within the meaning of polypeptide. The terms alsoinclude molecules in which one or more amino acid analogs ornon-canonical or unnatural amino acids are included as can be expressedrecombinantly using known protein engineering techniques. In addition,fusion proteins can be derivatized as described herein by well-knownorganic chemistry techniques.

A “variant” of a polypeptide (e.g., an immunoglobulin, or an antibody)comprises an amino acid sequence wherein one or more amino acid residuesare inserted into, deleted from and/or substituted into the amino acidsequence relative to another polypeptide sequence. Variants includefusion proteins.

The term “fusion protein,” for example with respect to aflibercept,indicates that the protein includes polypeptide components derived frommore than one parental protein or polypeptide. Typically, a fusionprotein is expressed from a “fusion gene” in which a nucleotide sequenceencoding a polypeptide sequence from one protein is appended in framewith, and optionally separated by a linker from, a nucleotide sequenceencoding a polypeptide sequence from a different protein. The fusiongene can then be expressed by a recombinant host cell as a singleprotein.

A “secreted” protein refers to those proteins capable of being directedto the endoplasmic reticulum (ER), secretory vesicles, or theextracellular space as a result of a secretory signal peptide sequence,as well as those proteins released into the extracellular space withoutnecessarily containing a signal sequence. If the secreted protein isreleased into the extracellular space, the secreted protein can undergoextracellular processing to produce a “mature” protein. Release into theextracellular space can occur by many mechanisms, including exocytosisand proteolytic cleavage. In some other embodiments, the afliberceptfusion protein of interest can be synthesized by the host cell as asecreted protein, which can then be further purified from theextracellular space and/or medium.

As used herein “soluble” when in reference to a protein produced byrecombinant DNA technology in a host cell is a protein that exists inaqueous solution; if the protein contains a twin-arginine signal aminoacid sequence the soluble protein is exported to the periplasmic spacein gram negative bacterial hosts, or is secreted into the culture mediumby eukaryotic host cells capable of secretion, or by bacterial hostpossessing the appropriate genes (e.g., the kil gene). Thus, a solubleprotein is a protein which is not found in an inclusion body inside thehost cell. Alternatively, depending on the context, a soluble protein isa protein which is not found integrated in cellular membranes, or, invitro, is dissolved, or is capable of being dissolved in an aqueousbuffer under physiological conditions without forming significantamounts of insoluble aggregates (i.e., forms aggregates less than 10%,and typically less than about 5%, of total protein) when it is suspendedwithout other proteins in an aqueous buffer of interest underphysiological conditions, such buffer not containing an ionic detergentor chaotropic agent, such as sodium dodecyl sulfate (SDS), urea,guanidinium hydrochloride, or lithium perchlorate. In contrast, aninsoluble protein is one which exists in denatured form insidecytoplasmic granules (called an inclusion body) in the host cell, oragain depending on the context, an insoluble protein is one which ispresent in cell membranes, including but not limited to, cytoplasmicmembranes, mitochondrial membranes, chloroplast membranes, endoplasmicreticulum membranes, etc., or in an in vitro aqueous buffer underphysiological conditions forms significant amounts of insolubleaggregates (i.e., forms aggregates equal to or more than about 10% oftotal protein) when it is suspended without other proteins (atphysiologically compatible temperature) in an aqueous buffer of interestunder physiological conditions, such buffer not containing an ionicdetergent or chaotropic agent, such as sodium dodecyl sulfate (SDS),urea, guanidinium hydrochloride, or lithium perchlorate.

The term “polynucleotide” or “nucleic acid” includes bothsingle-stranded and double-stranded nucleotide polymers containing twoor more nucleotide residues. The nucleotide residues comprising thepolynucleotide can be ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. Said modifications includebase modifications such as bromouridine and inosine derivatives, ribosemodifications such as 2′,3′-dideoxyribose, and internucleotide linkagemodifications such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotide residues. In some embodiments, oligonucleotides are 10to 60 bases in length. In other embodiments, oligonucleotides are 12,13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length.Oligonucleotides may be single stranded or double stranded, e.g., foruse in the construction of a mutant gene. Oligonucleotides may be senseor antisense oligonucleotides. An oligonucleotide can include a label,including a radiolabel, a fluorescent label, a hapten or an antigeniclabel, for detection assays. Oligonucleotides may be used, for example,as PCR primers, cloning primers or hybridization probes.

A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acidsequence,” as used interchangeably herein, is the primary sequence ofnucleotide residues in a polynucleotide, including of anoligonucleotide, a DNA, and RNA, a nucleic acid, or a character stringrepresenting the primary sequence of nucleotide residues, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence can bedetermined. Included are DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Unless specified otherwise, the left-hand end of anysingle-stranded polynucleotide sequence discussed herein is the 5′ end;the left-hand direction of double-stranded polynucleotide sequences isreferred to as the 5′ direction. The direction of 5′ to 3′ addition ofnascent RNA transcripts is referred to as the transcription direction;sequence regions on the DNA strand having the same sequence as the RNAtranscript that are 5′ to the 5′ end of the RNA transcript are referredto as “upstream sequences;” sequence regions on the DNA strand havingthe same sequence as the RNA transcript that are 3′ to the 3′ end of theRNA transcript are referred to as “downstream sequences.”

As used herein, an “isolated nucleic acid molecule” or “isolated nucleicacid sequence” is a nucleic acid molecule that is either (1) identifiedand separated from at least one contaminant nucleic acid molecule withwhich it is ordinarily associated in the natural source of the nucleicacid or (2) cloned, amplified, tagged, or otherwise distinguished frombackground nucleic acids such that the sequence of the nucleic acid ofinterest can be determined. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the immunoglobulin (e.g.,antibody) where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of ribonucleotidesalong the mRNA chain, and also determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for the RNAsequence and for the amino acid sequence.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Genes typically include coding sequencesand/or the regulatory sequences required for expression of such codingsequences. The term “gene” applies to a specific genomic or recombinantsequence, as well as to a cDNA or mRNA encoded by that sequence. Genesalso include non-expressed nucleic acid segments that, for example, formrecognition sequences for other proteins. Non-expressed regulatorysequences including transcriptional control elements to which regulatoryproteins, such as transcription factors, bind, resulting intranscription of adjacent or nearby sequences.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent post-translational modification of thepolypeptide), or both transcription and translation, as indicated by thecontext.

An expression cassette is a typical feature of recombinant expressiontechnology. The expression cassette includes a gene encoding a proteinof interest, e.g., a gene encoding an aflibercept fusion proteinsequence. A eukaryotic “expression cassette” refers to the part of anexpression vector that enables production of protein in a eukaryoticcell, such as a mammalian cell. It includes a promoter, operable in aeukaryotic cell, for mRNA transcription, one or more gene(s) encodingprotein(s) of interest and a mRNA termination and processing signal. Anexpression cassette can usefully include among the coding sequences, agene useful as a selective marker. In the expression cassette promoteris operably linked 5′ to an open reading frame encoding an exogenousprotein of interest; and a polyadenylation site is operably linked 3′ tothe open reading frame. Other suitable control sequences can also beincluded as long as the expression cassette remains operable. The openreading frame can optionally include a coding sequence for more than oneprotein of interest.

As used herein the term “coding region” or “coding sequence” when usedin reference to a structural gene refers to the nucleotide sequenceswhich encode the amino acids found in the nascent polypeptide as aresult of translation of an mRNA molecule. The coding region is bounded,in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” whichencodes the initiator methionine and on the 3′ side by one of the threetriplets which specify stop codons (i.e., TAA, TAG, TGA).

Recombinant expression technology typically involves the use of arecombinant expression vector comprising an expression cassette.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” as used hereinrefers to a recombinant DNA molecule containing a desired codingsequence and appropriate nucleic acid control sequences necessary forthe expression of the operably linked coding sequence in a particularhost cell. An expression vector can include, but is not limited to,sequences that affect or control transcription, translation, and, ifintrons are present, affect RNA splicing of a coding region operablylinked thereto. Nucleic acid sequences necessary for expression inprokaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals. A secretory signal peptide sequence can also,optionally, be encoded by the expression vector, operably linked to thecoding sequence of interest, so that the expressed polypeptide can besecreted by the recombinant host cell, for more facile isolation of thepolypeptide of interest from the cell, if desired. Such techniques arewell known in the art. (E.g., Goodey, Andrew R.; et al., Peptide and DNAsequences, U.S. Pat. No. 5,302,697; Weiner et al., Compositions andmethods for protein secretion, U.S. Pat. Nos. 6,022,952 and 6,335,178;Uemura et al., Protein expression vector and utilization thereof, U.S.Pat. No. 7,029,909; Ruben et al., 27 human secreted proteins, US2003/0104400 A1). For expression of multi-subunit proteins of interest,separate expression vectors in suitable numbers and proportions, eachcontaining a coding sequence for each of the different subunit monomers,can be used to transform a host cell. In other embodiments, a singleexpression vector can be used to express the different subunits of theprotein of interest.

Recombinant expression technology typically involves a mammalian hostcell comprising the recombinant expression vector.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid and thereby expressesa gene or coding sequence of interest. The term includes the progeny ofthe parent cell, whether or not the progeny is identical in morphologyor in genetic make-up to the original parent cell, so long as the geneof interest is present. Any of a large number of available andwell-known host cells may be used in the practice of this invention toobtain aflibercept. The selection of a particular host is dependent upona number of factors recognized by the art. These include, for example,compatibility with the chosen expression vector, toxicity of thepeptides encoded by the DNA molecule, rate of transformation, ease ofrecovery of the peptides, expression characteristics, bio-safety andcosts. A balance of these factors must be struck with the understandingthat not all hosts may be equally effective for the expression of aparticular DNA sequence. Within these general guidelines, usefulmicrobial host cells in culture include bacteria (such as Escherichiacoli sp.), yeast (such as Saccharomyces sp.) and other fungal cells,algal or algal-like cells, insect cells, plant cells, mammalian(including human) cells, e.g., CHO cells and HEK-293 cells.Modifications can be made at the DNA level, as well. Thepeptide-encoding DNA sequence may be changed to codons more compatiblewith the chosen host cell. For E. coli, optimized codons are known inthe art. Codons can be substituted to eliminate restriction sites or toinclude silent restriction sites, which may aid in processing of the DNAin the selected host cell. Next, the transformed host is cultured andpurified. Host cells may be cultured under conventional fermentationconditions so that the desired compounds are expressed. Suchfermentation conditions are well known in the art.

Examples of useful mammalian host cell lines are Chinese hamster ovarycells, including CHO-K1 cells (e.g., ATCC CCL61), DXB-11, DG-44, andChinese hamster ovary cells/−DHFR (CHO, Urlaub et al, Proc. Natl. Acad.Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture (Graham et al, J. Gen Virol.36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouseSertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkeykidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanhepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383: 44-68(1982)); MRC 5 cells or FS4 cells; or mammalian myeloma cells.

“Cell,” “cell line,” and “cell culture” are often used interchangeablyand all such designations herein include cellular progeny. For example,a cell “derived” from a CHO cell is a cellular progeny of a ChineseHamster Ovary cell, which may be removed from the original primary cellparent by any number of generations, and which can also include atransformant progeny cell. Transformants and transformed cells includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included.

Host cells are transformed or transfected with the above-describednucleic acids or vectors for production of polypeptides (includingantigen binding proteins, such as antibodies) and are cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful for the expression of polypeptides, suchas antibodies.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAmay recombine with that of the cell by physically integrating into achromosome of the cell, or may be maintained transiently as an episomalelement without being replicated, or may replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

The host cells used to produce the aflibercept fusion polypeptidesuseful in the invention may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;WO90103430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source, such that the physiologicalconditions of the cell in, or on, the medium promote expression of theprotein of interest by the host cell; any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such as temperature(typically, but not necessarily, about 37° C.), pH (typically, but notnecessarily, about pH 6.5-7.5), oxygenation, and the like, are thosepreviously used with the host cell selected for expression of theprotein of interest, and will be apparent to the ordinarily skilledartisan. The culture medium can include a suitable amount of serum sucha fetal bovine serum (FBS), or preferably, the host cells can be adaptedfor culture in serum-free medium. In some embodiments, the aqueousmedium is liquid, such that the host cells are cultured in a cellsuspension within the liquid medium. The host cells can be usefullygrown in batch culture or in continuous culture systems.

In other embodiments, the mammalian host cells can be cultured on solidor semi-solid aqueous medium, for example, containing agar or agarose,to form a medium or substrate surface to which the cells adhere and forman adhesion layer.

Upon culturing the host cells, the recombinant polypeptide can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the polypeptide, such as aflibercept, is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration.

A protein of interest, such as aflibercept, can be purified using, forexample, hydroxylapatite chromatography, cation or anion exchangechromatography, or preferably affinity chromatography, using the antigenof interest or protein A or protein G as an affinity ligand. Protein Acan be used to purify proteins that include polypeptides are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al, EMBO J. 5: 15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe protein comprises a CH 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as ethanol precipitation, Reverse PhaseHPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation arealso possible depending on the antibody to be recovered.

“Under physiological conditions” with respect to incubating buffers andimmunoglobulins, or other binding assay reagents means incubation underconditions of temperature, pH, and ionic strength, that permit abiochemical reaction, such as a non-covalent binding reaction, to occur.Typically, the temperature is at room or ambient temperature up to about37° C. and at pH 6.5-7.5.

“Physiologically acceptable salt” of a composition of matter, forexample a salt of a protein of interest, e.g., a fusion protein or animmunoglobulin, such as an antibody, or any other protein of interest,or a salt of an amino acid, such as, but not limited to, a lysine,histidine, or proline salt, means any salt, or salts, that are known orlater discovered to be pharmaceutically acceptable. Some non-limitingexamples of pharmaceutically acceptable salts are: acetate salts;trifluoroacetate salts; hydrohalides, such as hydrochloride (e.g.,monohydrochloride or dihydrochloride salts) and hydrobromide salts;sulfate salts; citrate salts; maleate salts; tartrate salts; glycolatesalts; gluconate salts; succinate salts; mesylate salts; besylate salts;salts of gallic acid esters (gallic acid is also known as 3,4, 5trihydroxybenzoic acid) such as PentaGalloylGlucose (PGG) andepigallocatechin gallate (EGCG), salts of cholesteryl sulfate, pamoatesalts, tannate salts, and oxalate salts.

A “reaction mixture” is an aqueous mixture containing all the reagentsand factors necessary, which under physiological conditions ofincubation, permit an in vitro biochemical reaction of interest tooccur, such as a covalent or non-covalent binding reaction.

A “domain” or “region” (used interchangeably herein) of a polynucleotideis any portion of the entire polynucleotide, up to and including thecomplete polynucleotide, but typically comprising less than the completepolynucleotide. A domain can, but need not, fold independently (e.g.,DNA hairpin folding) of the rest of the polynucleotide chain and/or becorrelated with a particular biological, biochemical, or structuralfunction or location, such as a coding region or a regulatory region.

A “domain” or “region” (used interchangeably herein) of a protein is anyportion of the entire protein, up to and including the complete protein,but typically comprising less than the complete protein. A domain can,but need not, fold independently of the rest of the protein chain and/orbe correlated with a particular biological, biochemical, or structuralfunction or location (e.g., a ligand binding domain, or a cytosolic,transmembrane or extracellular domain).

Quantification of aflibercept fusion protein, is often useful ornecessary in tracking protein production or for lot release assays ofdrug substance or drug product containing aflibercept. An antibody thatspecifically binds aflibercept, particularly a monoclonal antibody, cantherefore be useful for these purposes.

The term “antibody”, or interchangeably “Ab”, is used in the broadestsense and includes fully assembled antibodies, monoclonal antibodies(including human, humanized or chimeric antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments that can bind antigen (e.g., Fab, Fab′, F(ab′)2, Fv,single chain antibodies, diabodies), comprising complementaritydetermining regions (CDRs) of the foregoing as long as they exhibit thedesired biological activity. Multimers or aggregates of intact moleculesand/or fragments, including chemically derivatized antibodies, arecontemplated. Antibodies of any isotype class or subclass, includingIgG, IgM, IgD, IgA, and IgE, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, orany allotype, are contemplated. Different isotypes have differenteffector functions; for example, IgG1 and IgG3 isotypes haveantibody-dependent cellular cytotoxicity (ADCC) activity.

An “isolated” protein, e.g., an aflibercept fusion protein, is one thathas been identified and separated from one or more components of itsnatural environment or of a culture medium in which it has been secretedby a producing cell. In some embodiments, the isolated protein issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural or culture medium environment that wouldinterfere with its therapeutic, diagnostic, prophylactic, research orother use. “Contaminant” components of its natural environment or mediumare materials that would interfere with diagnostic or therapeutic usesfor the protein, e.g., an antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous (e.g., polynucleotides,lipids, carbohydrates) solutes. Typically, an “isolated protein”constitutes at least about 5%, at least about 10%, at least about 25%,or at least about 50% of a given sample. In some embodiments, theprotein of interest, e.g., aflibercept fusion protein or an antibody,will be purified (1) to greater than 95% by weight of protein, and mostpreferably more than 99% by weight, or (2) to homogeneity by SDS-PAGE,or other suitable technique, under reducing or nonreducing conditions,optionally using a stain, e.g., Coomassie blue or silver stain. Isolatednaturally occurring antibody includes the antibody in situ withinrecombinant cells since at least one component of the protein's naturalenvironment will not be present. Typically, however, the isolatedprotein of interest (e.g., aflibercept or an antibody) will be preparedby at least one purification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies that are antigen binding proteinsare highly specific binders, being directed against an individualantigenic site or epitope, in contrast to polyclonal antibodypreparations that typically include different antibodies directedagainst different epitopes. Nonlimiting examples of monoclonalantibodies include murine, rabbit, rat, chicken, chimeric, humanized, orhuman antibodies, fully assembled antibodies, multispecific antibodies(including bispecific antibodies), antibody fragments that can bind anantigen (including, Fab, Fab′, F(ab)₂, Fv, single chain antibodies,diabodies), maxibodies, nanobodies, and recombinant peptides comprisingCDRs of the foregoing as long as they exhibit the desired biologicalactivity, or variants or derivatives thereof.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiesmay be made by the hybridoma method first described by Kohler et al.,Nature, 256:495 (1975), or may be made by recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

The term “immunoglobulin” encompasses full antibodies comprising twodimerized heavy chains (HC), each covalently linked to a light chain(LC); a single undimerized immunoglobulin heavy chain and covalentlylinked light chain (HC+LC), or a chimeric immunoglobulin (lightchain+heavy chain)-Fc heterotrimer (a so-called “hemibody”). An“immunoglobulin” is a protein, but is not necessarily an antigen bindingprotein.

In an “antibody”, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” chain of about 220amino acids (about 25 kDa) and one “heavy” chain of about 440 aminoacids (about 50-70 kDa). The amino-terminal portion of each chainincludes a “variable” (“V”) region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. The variable region differsamong different antibodies. The constant region is the same amongdifferent antibodies. Within the variable region of each heavy or lightchain, there are three hypervariable subregions that help determine theantibody's specificity for antigen in the case of an antibody that is anantigen binding protein. The variable domain residues between thehypervariable regions are called the framework residues and generallyare somewhat homologous among different antibodies Immunoglobulins canbe assigned to different classes depending on the amino acid sequence ofthe constant domain of their heavy chains. Human light chains areclassified as kappa (.kappa.) and lambda (.lamda.) light chains. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). An “antibody” also encompasses a recombinantly made antibody,and antibodies that are glycosylated or lacking glycosylation.

The term “light chain” or “immunoglobulin light chain” includes afull-length light chain and fragments thereof having sufficient variableregion sequence to confer binding specificity. A full-length light chainincludes a variable region domain, V_(L), and a constant region domain,C_(L). The variable region domain of the light chain is at theamino-terminus of the polypeptide. Light chains include kappa chains andlambda chains.

The term “heavy chain” or “immunoglobulin heavy chain” includes afull-length heavy chain and fragments thereof having sufficient variableregion sequence to confer binding specificity. A full-length heavy chainincludes a variable region domain, V_(H), and three constant regiondomains, C_(H1), C_(H2), and C_(H3). The V_(H) domain is at theamino-terminus of the polypeptide, and the C_(H) domains are at thecarboxyl-terminus, with the C_(H3) being closest to the carboxy-terminusof the polypeptide. Heavy chains are classified as mu (μ), delta (δ),gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotypeas IgM, IgD, IgG, IgA, and IgE, respectively. Heavy chains may be of anyisotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes),IgA (including IgA1 and IgA2 subtypes), IgM and IgE. Several of thesemay be further divided into subclasses or isotypes, e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2. Different IgG isotypes may have differenteffector functions (mediated by the Fc region), such asantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fcreceptors (Fc.gamma.Rs) on the surface of immune effector cells such asnatural killers and macrophages, leading to the phagocytosis or lysis ofthe targeted cells. In CDC, the antibodies kill the targeted cells bytriggering the complement cascade at the cell surface.

An “Fc region”, or used interchangeably herein, “Fc domain” or“immunoglobulin Fc domain”, contains two heavy chain fragments, which ina full antibody comprise the C_(H1) and C_(H2) domains of the antibody.The two heavy chain fragments are held together by two or more disulfidebonds and by hydrophobic interactions of the C_(H3) domains.

The term “salvage receptor binding epitope” refers to an epitope of theFc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

For a detailed description of the structure and generation ofantibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414 (1998),herein incorporated by reference in its entirety. Briefly, the processfor generating DNA encoding the heavy and light chain immunoglobulinsequences occurs primarily in developing B-cells. Prior to therearranging and joining of various immunoglobulin gene segments, the V,D, J and constant (C) gene segments are found generally in relativelyclose proximity on a single chromosome. During B-cell-differentiation,one of each of the appropriate family members of the V, D, J (or only Vand J in the case of light chain genes) gene segments are recombined toform functionally rearranged variable regions of the heavy and lightimmunoglobulin genes. This gene segment rearrangement process appears tobe sequential. First, heavy chain D-to-J joints are made, followed byheavy chain V-to-DJ joints and light chain V-to-J joints. In addition tothe rearrangement of V, D and J segments, further diversity is generatedin the primary repertoire of immunoglobulin heavy and light chains byway of variable recombination at the locations where the V and Jsegments in the light chain are joined and where the D and J segments ofthe heavy chain are joined. Such variation in the light chain typicallyoccurs within the last codon of the V gene segment and the first codonof the J segment. Similar imprecision in joining occurs on the heavychain chromosome between the D and J_(H) segments and may extend over asmany as 10 nucleotides. Furthermore, several nucleotides may be insertedbetween the D and J_(H) and between the V_(H) and D gene segments whichare not encoded by genomic DNA. The addition of these nucleotides isknown as N-region diversity. The net effect of such rearrangements inthe variable region gene segments and the variable recombination whichmay occur during such joining is the production of a primary antibodyrepertoire.

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a complementarity determiningregion or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)]. Even asingle CDR may recognize and bind antigen, although with a loweraffinity than the entire antigen binding site containing all of theCDRs.

An alternative definition of residues from a hypervariable “loop” isdescribed by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) asresidues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain.

“Framework” or “FR” residues are those variable region residues otherthan the hypervariable region residues.

“Antibody fragments” comprise a portion of an intact full lengthantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng., 8(10):1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment which contains the constant region.The Fab fragment contains all of the variable domain, as well as theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. The Fc fragment displays carbohydrates and isresponsible for many antibody effector functions (such as bindingcomplement and cell receptors), that distinguish one class of antibodyfrom another.

Pepsin treatment yields an F(ab′)₂ fragment that has two “Single-chainFv” or “scFv” antibody fragments comprising the V_(H) and V_(L) domainsof antibody, wherein these domains are present in a single polypeptidechain. Fab fragments differ from Fab′ fragments by the inclusion of afew additional residues at the carboxy terminus of the heavy chain CH1domain including one or more cysteines from the antibody hinge region.Preferably, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the Fv to form thedesired structure for antigen binding. For a review of scFv seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the CH1 domain and also theregion between the CH1 and CH2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the VH VL dimer. A single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203, the disclosures of which are incorporated by reference intheir entireties.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain, and optionally comprising a polypeptide linkerbetween the VH and VL domains that enables the Fv to form the desiredstructure for antigen binding (Bird et al., Science 242:423-426, 1988,and Huston et al., Proc. Nati. Acad. Sci. USA 85:5879-5883, 1988). An“Fd” fragment consists of the V_(H) and C_(H1) domains.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

The term “antigen binding protein” (ABP) includes aflibercept, orantibodies or antibody fragments, as defined herein, and recombinantpeptides or other compounds that contain sequences derived from CDRshaving the desired antigen-binding properties such that theyspecifically bind a target antigen of interest.

In general, an antigen binding protein, e.g., aflibercept or an antibodyor antibody fragment, “specifically binds” to an antigen of interestwhen it has a significantly higher binding affinity for, andconsequently is capable of distinguishing, that antigen, compared to itsaffinity for other unrelated proteins, under similar binding assayconditions. Typically, an antigen binding protein is said to“specifically bind” its target antigen when the dissociation constant(K_(D)) is 10⁻⁸ M or lower. The antigen binding protein specificallybinds antigen with “high affinity” when the K_(D) is 10⁻⁹ M or lower,and with “very high affinity” when the K_(D) is 10⁻¹⁰ M or lower.

“Antigen binding region” or “antigen binding site” means a portion of aprotein that specifically binds a specified antigen. For example, thatportion of an antigen binding protein that contains the amino acidresidues that interact with an antigen and confer on the antigen bindingprotein its specificity and affinity for the antigen is referred to as“antigen binding region.” An antigen binding region typically includesone or more “complementary binding regions” (“CDRs”). Certain antigenbinding regions also include one or more “framework” regions (“FRs”). A“CDR” is an amino acid sequence that contributes to antigen bindingspecificity and affinity. “Framework” regions can aid in maintaining theproper conformation of the CDRs to promote binding between the antigenbinding region and an antigen. In a traditional antibody, the CDRs areembedded within a framework in the heavy and light chain variable regionwhere they constitute the regions responsible for antigen binding andrecognition. A variable region of an immunoglobulin antigen bindingprotein comprises at least three heavy or light chain CDRs, see, supra(Kabat et al., 1991, Sequences of Proteins of Immunological Interest,Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk,1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), within a framework region (designated framework regions 1-4,FR1, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see also Chothiaand Lesk, 1987, supra).

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as an antigenbinding protein (including, e.g., aflibercept, or an antibody orimmunologically functional fragment of an antibody), and additionallycapable of being used in an animal to produce antibodies capable ofbinding to that antigen. An antigen may possess one or more epitopesthat are capable of interacting with different antigen binding proteins,e.g., antibodies.

The term “epitope” is the portion of a molecule that is bound by anantigen binding protein (for example, aflibercept or an antibody). Theterm includes any determinant capable of specifically binding to anantigen binding protein, such as an antibody or to a T-cell receptor. Anepitope can be contiguous or non-contiguous (e.g., in a single-chainpolypeptide, amino acid residues that are not contiguous to one anotherin the polypeptide sequence but that within the context of the moleculeare bound by the antigen binding protein). In certain embodiments,epitopes may be mimetic in that they comprise a three dimensionalstructure that is similar to an epitope used to generate the antigenbinding protein, yet comprise none or only some of the amino acidresidues found in that epitope used to generate the antigen bindingprotein. Most often, epitopes reside on proteins, but in some instancesmay reside on other kinds of molecules, such as nucleic acids. Epitopedeterminants may include chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl or sulfonylgroups, and may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. Generally,antibodies specific for a particular target antigen will preferentiallyrecognize an epitope on the target antigen in a complex mixture ofproteins and/or macromolecules.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.For example, sequence identity can be determined by standard methodsthat are commonly used to compare the similarity in position of theamino acids of two polypeptides. Using a computer program such as BLASTor FASTA, two polypeptide or two polynucleotide sequences are alignedfor optimal matching of their respective residues (either along the fulllength of one or both sequences, or along a pre-determined portion ofone or both sequences). The programs provide a default opening penaltyand a default gap penalty, and a scoring matrix such as PAM 250 [astandard scoring matrix; see Dayhoff et al., in Atlas of ProteinSequence and Structure, vol. 5, supp. 3 (1978)] can be used inconjunction with the computer program. For example, the percent identitycan then be calculated as: the total number of identical matchesmultiplied by 100 and then divided by the sum of the length of thelonger sequence within the matched span and the number of gapsintroduced into the longer sequences in order to align the twosequences. In calculating percent identity, the sequences being comparedare aligned in a way that gives the largest match between the sequences.

The GCG program package is a computer program that can be used todetermine percent identity, which package includes GAP (Devereux et al.,1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University ofWisconsin, Madison, Wis.). The computer algorithm GAP is used to alignthe two polypeptides or two polynucleotides for which the percentsequence identity is to be determined. The sequences are aligned foroptimal matching of their respective amino acid or nucleotide (the“matched span”, as determined by the algorithm). A gap opening penalty(which is calculated as 3.times. the average diagonal, wherein the“average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 1/10 times the gap openingpenalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62are used in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see, Dayhoff et al., 1978, Atlas of ProteinSequence and Structure 5:345-352 for the PAM 250 comparison matrix;Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 forthe BLOSUM 62 comparison matrix) is also used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program include the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 contiguous amino acids of the target polypeptide.

The term “modification” when used in connection with proteins ofinterest, include, but are not limited to, one or more amino acidchanges (including substitutions, insertions or deletions); chemicalmodifications; covalent modification by conjugation to therapeutic ordiagnostic agents; labeling (e.g., with radionuclides or variousenzymes); covalent polymer attachment such as PEGylation (derivatizationwith polyethylene glycol) and insertion or substitution by chemicalsynthesis of non-natural amino acids. By methods known to the skilledartisan, proteins, can be “engineered” or modified for improved targetaffinity, selectivity, stability, and/or manufacturability before thecoding sequence of the “engineered” protein is included in theexpression cassette.

The term “derivative” when used in connection with proteins of interest,such as aflibercept or antibodies, refers to proteins that arecovalently modified by conjugation to therapeutic or diagnostic agents,labeling (e.g., with radionuclides or various enzymes), covalent polymerattachment such as PEGylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of non-natural aminoacids.

Within the scope of the invention, aflibercept proteins can betherapeutic proteins, or “biologics,” for the treatment of disease,including but not limited to human disease or disorder, e.g., a diseaseor disorder of the eye. “Treatment” or “treating” is an interventionperformed with the intention of preventing the development or alteringthe pathology of a disorder. Accordingly, “treatment” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. “Treatment” includes anyindication(s) of success in the amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examinationby a physician, e.g., an ophthalmologist, or other health care provider,or self-reporting by a patient.

An “effective amount” of a therapeutic is generally an amount sufficientto reduce the severity and/or frequency of symptoms, eliminate thesymptoms and/or underlying cause, prevent the occurrence of symptomsand/or their underlying cause, and/or improve or remediate the damagethat results from or is associated with an eye disorder or disease. Insome embodiments, the effective amount is a therapeutically effectiveamount or a prophylactically effective amount. A “therapeuticallyeffective amount” is an amount sufficient to remedy a disease state(e.g., macular edema following Central Retinal Vein Occlusion (CRVO),Central Retinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion(BRVO), Neovascular (Wet) Age-Related Macular Degeneration (AMD),Impaired vision due to Myopic Choroidal Neovascularisation, DiabeticMacular Edema (DME), Diabetic Retinopathy (DR) in patients with DME, andneovascular Age-Related Macular Degeneration (AMD), transplant rejectionor GVHD, inflammation, multiple sclerosis, cancer, cardiovasculardisease, diabetes, neuropathy, pain) or symptom(s), particularly a stateor symptom(s) associated with the disease state, or otherwise prevent,hinder, retard or reverse the progression of the disease state or anyother undesirable symptom associated with the disease in any waywhatsoever (i.e. that provides “therapeutic efficacy”). A“prophylactically effective amount” is an amount of a pharmaceuticalcomposition that, when administered to a subject, will have the intendedprophylactic effect. The full therapeutic or prophylactic effect doesnot necessarily occur by administration of one dose, and may occur onlyafter administration of a series of doses. Thus, a therapeutically orprophylactically effective amount may be administered in one or moreadministrations.

Cloning DNA

Cloning of DNA is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, which is incorporated herein by reference).For example, a cDNA library may be constructed by reverse transcriptionof polyA+ mRNA, preferably membrane-associated mRNA, and the libraryscreened using probes specific for human immunoglobulin polypeptide genesequences. In one embodiment, however, the polymerase chain reaction(PCR) is used to amplify cDNAs (or portions of full-length cDNAs)encoding an immunoglobulin gene segment of interest (e.g., a light orheavy chain variable segment). The amplified sequences can be readilycloned into any suitable vector, e.g., expression vectors, minigenevectors, or phage display vectors. It will be appreciated that theparticular method of cloning used is not critical, so long as it ispossible to determine the sequence of some portion of the polypeptide ofinterest, e.g., of the aflibercept fusion polypeptide sequence.

One source for antibody nucleic acids is a hybridoma produced byobtaining a B cell from an animal immunized with the antigen of interestand fusing it to an immortal cell. Alternatively, nucleic acid can beisolated from B cells (or whole spleen) of the immunized animal. Yetanother source of nucleic acids encoding antibodies is a library of suchnucleic acids generated, for example, through phage display technology.Polynucleotides encoding peptides of interest, e.g., variable regionpeptides with desired binding characteristics, can be identified bystandard techniques such as panning.

Sequencing of DNA is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad.Sci. USA 74: 5463-5467, which is incorporated herein by reference). Bycomparing the sequence of the cloned nucleic acid with publishedsequences of genes and cDNAs, one of skill will readily be able todetermine, depending on the region sequenced. One source of genesequence information is the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md.

Isolated DNA can be operably linked to control sequences or placed intoexpression vectors, which are then transfected into host cells that donot otherwise produce immunoglobulin protein, to direct the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantproduction of antibodies is well known in the art.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Many vectors are known in the art. Vector components may include one ormore of the following: a signal sequence (that may, for example, directsecretion of the expressed protein; an origin of replication, one ormore selective marker genes (that may, for example, confer antibiotic orother drug resistance, complement auxotrophic deficiencies, or supplycritical nutrients not available in the media), an enhancer element, apromoter, and a transcription termination sequence, all of which arewell known in the art.

Purity of Water and other Ingredients. The water and all otheringredients that are used to make the inventive ophthalmic formulationsare preferably of a level of purity meeting the applicable legal orpharmacopoeial standards required for such pharmaceutical compositionsand medicaments in the jurisdiction of interest, e.g., United StatesPharmacopeia (USP), European Pharmacopeia, Japanese Pharmacopeia, orChinese Pharmacopeia, etc. For example, according to the USP, Water forInjection is used as an excipient in the production of parenteral andother preparations where product endotoxin content must be controlled,and in other pharmaceutical applications, such as cleaning of certainequipment and parenteral product-contact components; and the minimumquality of source or feed water for the generation of Water forInjection is Drinking Water as defined by the U.S. EnvironmentalProtection Agency (EPA), EU, Japan, or WHO.

Before administration to a patient, the inventive formulations shouldmeet the applicable legal or pharmacopoeial standards required for suchpharmaceutical compositions and medicaments in the jurisdiction ofinterest as to sterility, lack of endotoxin or viral contaminants, etc.

Buffer Systems

The ophthalmic formulation of the invention includes a buffer in therange of about 5 to 50 mM concentration. A suitable buffer system forthe inventive ophthalmic formulation can be chosen from a phosphatebuffer, histidine buffer, acetate buffer, succinate buffer, citratebuffer, glutamate, and lactate, or the buffer can be a combination oftwo or more of these buffer systems. Some useful embodiments of theinvention have a buffer concentration in the range of about 5 mM toabout 20 mM, and other embodiments have a buffer concentration of about5 to about 10 mM. If a histidine buffer is selected, a histidineconcentration in the range of about 5-20 mM is preferred.

Non-Ionic Surfactants

The inventive ophthalmic formulation includes a non-ionic surfactant,preferably at a concentration of about 0.001% (w/v) to about 5.0% (w/v).In some embodiments the concentration of the non-ionic surfactant isabout 0.001% (w/v) to about 2.0% (w/v), or about 0.001% (w/v) to about1.0% (w/v), or about 0.001% (w/v) to about 0.10% (w/v), or about 0.001%(w/v) to about 0.01% (w/v). A useful non-ionic surfactant can be apolysorbate (e.g., polysorbate 20 or polysorbate 80), Brij 35 (i.e.,polyethylene glycol dodecyl ether), a poloxamer (i.e.,Polyethylene-Polypropylene Glycol; Polyoxyethylene-PolyoxypropyleneBlock Copolymer; Poly(Ethylene oxide-co-Polypropylene oxide)) such asPoloxamer 188 (i.e., Pluronic F68), or Triton™ X-100 (i.e.,4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol)). Alsoencompassed within “non-ionic surfactant” for purposes of practicing thepresent invention are alkylsaccharides or alkylglycosides (e.g., soldunder the trade name ProTek® by Aegis Therapeutics, LLC; see, e.g.,Maggio, Stabilizing Alkylglycoside Compositions And Methods Thereof,U.S. Pat. No. 8,133,863 B2).

Tonicifying Agents

The inventive ophthalmic formulation includes a tonicifying agent suchthat the formulation has a final osmolality of about 300 mOsm/kg (i.e.,300±50 mOsm/kg) and the chloride anion concentration is less than about10 mM, preferably less than about 5 mM, and more preferably less thanabout 1 mM. Osmolality is a measure of the number of dissolved particlesper unit of water. In a solution, the fewer the number of particles ofsolute in proportion to the number of units of water (solvent), the lessconcentrated the solution, hypo-osmotic. If a semi-permeable membrane(one that is permeable only to solvent molecules) is used to separatesolutions of different solute concentrations, a phenomenon known asosmosis occurs in which solvent molecules cross the membrane from lowerto higher concentration to establish a concentration equilibrium. Thepressure driving this movement is called osmotic pressure and isgoverned by the number of “particles” of solute in solution. Solutionscontaining the same concentration of particles and thus exerting equalosmotic pressures are called iso-osmotic. For example, the osmoticpressure within a red blood cell (rbc) is equal to the surroundingsolution so that it neither shrinks or expands. If a rbc is placed inwater it will burst since the water alone is hypo-osmotic. If the rbc isplaced in a high salt solution, i.e. greater than 0.9% (w/v) sodiumchloride, it will shrink since the solution is hyper-osmotic. In bothexamples the rbc is damaged. The same will happen with any biologicalcell, such as those within the eye. If a hypo- or hyper-osmotic solutionis placed on the eye it will cause damage, thus necessitating the needfor an iso-osmotic solution for drugs used for the eye. In practice, a0.9% (w/v) solution of sodium chloride is iso-osmotic and has aconcentration of 270-300 mOsm/kg. All solutions are compared to thisstandard and are considered iso-osmotic if they fall within the expandedrange of 250-350 mOsm/kg. Excipients used to stabilize proteins areadded at concentrations to produce iso-osmotic solutions. For example,disaccharides such as sucrose and trehalose are iso-osmotic atconcentrations of 9.25%, monosaccharides such as glucose and mannose areiso-osmotic at concentrations of 5%, and amino acids such as proline areiso-osmotic at concentrations of approximately 3%. Osmolality can bedetermined either theoretically or experimentally. Theoreticalcalculations can be determined according to the following equation:

Osmolality=(g compound/100 mL solution)*(Compound's E-value).

A compound's E-value is determined by the equation:

E-value=(MW NaCl/i-value NaCl)*(i-value compound/MW compound).

The i-value is the number of ions from a compound based on a theoreticaldissociation of 80%. For a compound that does not dissociate, i.e.sucrose, the i-value is 1. For a compound that dissociates into 2 ions,i.e. NaCl, the i-value is 1.8 and for a compound that dissociates into 3ions the i-value is 2.6. Since osmolality is a colligative property ofthe solution, depression of the freezing point due to added solutes or adepression of the vapor pressure are directly related to the totalnumber of solute molecules in a liquid. Each of these principles havebeen exploited in the art to develop useful instruments that measureosmolality. Either or both types of instruments can be used forbiological samples. As an example, a solution with 10 mM sodiumphosphate, 40 mM NaCl, 5% (w/v) sucrose and 0.03% (w/v) polysorbate 20would have a theoretical osmolality of 263 mOsm/kg. In our laboratory,the actual measured value was 270 mOsm/kg, as measured by freezing pointdepression. This illustrates that the experimental value closely matchesthe theoretical value, further demonstrating that for purposes ofpracticing the present invention, either a theoretical or experimentalvalue may be used for determining if a solution is suitable forintravitreal injection based on its osmolality.

A useful tonicifying agent can be a polyol or an amino acid. Examples ofuseful polyol tonicifying agents include sucrose, trehalose, sorbitol,mannitol, and glycerol. Typically, the concentration of the polyoltonicifying agent is the in the range of 5-10% (w/v), depending on thebuffer concentration and other formulation excipients. The concentrationof amino acid tonicifying agent is in the range of 2-4% (w/v), dependingon buffer concentration and other tonicifying agents used. In theinventive ophthalmic formulation, the amino acid tonicifying agent canbe a L-amino acid form and/or a D-amino acid form, if pharmaceuticallyacceptable. Encompassed also within the meaning of “amino acidtonicifying agent” is a pharmaceutically acceptable amino acid saltform.

Additional Stabilizing Agents

In some embodiments of the inventive ophthalmic formulation in which thetonicifying agent is a polyol, e.g., sucrose, trehalose, sorbitol,mannitol, or glycerol, the formulation also contains an additional aminoacid stabilizing agent. The additional amino acid stabilizing agent canbe, for example, proline, arginine, methionine, glycine, or lysine. Theadditional amino acid stabilizing agent can be an L-amino acid or aD-amino acid, or a salt form, as long as the amino acid ispharmaceutically acceptable, and in a pharmaceutically acceptable form,e.g., a pharmaceutically acceptable salt form. (See, e.g., Falconer etal., Stabilization of a monoclonal antibody during purification andformulation by addition of basic amino acid excipients, J Chem TechnolBiotechnol (2011) 86: 942-948; Platts et al., Control of GlobularProtein Thermal Stability in Aqueous Formulations by the PositivelyCharged Amino Acid Excipients, Journal of Pharmaceutical Sciences 105(2016) 3532-3536; Wang, W., Instability, stabilization, and formulationof liquid protein pharmaceuticals, International Journal ofPharmaceutics 185 (1999) 129-188; Yin et al., Effects of Antioxidants onthe Hydrogen Peroxide Mediated Oxidation of Methionine Residues inGranulocyte Colony-Stimulating Factor and Human Parathyroid HormoneFragment 13-34, Pharmaceutical Research (2004) 21(12):2377-2383; Lam etal., Antioxidants for Prevention of Methionine Oxidation in RecombinantMonoclonal Antibody HER2, Journal of Pharmaceutical Sciences 86(11):1250-1255 (1997); Levine et al., Methionine Residues as EndogenousAntioxidants in Proteins, Proc. Natl. Acad. Sci. (USA)93(26):15036-15040 (1996); Maeder et al., Local tolerance and stabilityup to 24 months of a new 20% proline-stabilized polyclonalimmunoglobulin for subcutaneous administration, Biologicals 39:43-49(2011); Cramer et al., Stability over 36 months of a new liquid 10%polyclonal immunoglobulin product (IgPro10, Privigen©) stabilized withL-proline, Vox Sanguinis (2009) 96, 219-225; Bolli et al., L-Prolinereduces IgG dimer content and enhances the stability of intravenousimmunoglobulin (IVIG) solutions, Biologicals 38 (2010) 150-157; Truong,Combination of D-Amino Acids and Lipoteichoic Acid, EP2545909 A1;Stroppolo et al., Pharmaceutical compositions containing the salts ofS(+)-2-(4-isobutylphenyl)propionic acid with basic aminoacids, U.S. Pat.No. 5,510,385). The concentration of the additional amino acidstabilizing agent in combination with the polyol is usefully about0.01-3% (w/v), depending on the concentration of polyol in combinationwith the amino acid. However, if methionine is selected in combinationwith a polyol tonicifying agent, methionine can be used as a scavengerof reactive oxygen species at a low concentration, i.e., 10 mM or less.

Exemplary Formulations of the Inventions

Exemplary ophthalmic formulations of the present invention include thosein which the buffer is a phosphate buffer. In one such embodiment (a)the aflibercept concentration is 20-80 mg/mL; (b) the phosphate bufferconcentration is about 10 mM; (c) the non-ionic surfactant ispolysorbate 20 at a concentration of about 0.03% (w/v); (d) thetonicifying agent is:

(i) sucrose or trehalose at a concentration of about 9% (w/v), or(ii) proline at a concentration of about 3% (w/v); (e) the concentrationof chloride anion is less than about 1 mM; and the pH of the formulationis about pH 6.0 to about pH 6.5. In some preferred embodiments of thisformulation, the tonicifying agent is sucrose or trehalose at aconcentration of about 9% (w/v), with the aflibercept concentration atabout 30 mg/mL to about 50 mg/mL; for example, a concentration of about40 mg/mL. In other preferred embodiments the tonicifying agent isproline at a concentration of about 3% (w/v), with the afliberceptconcentration at about 30 mg/mL to about 50 mg/mL; for example, aconcentration of about 40 mg/mL.

Exemplary ophthalmic formulations of the present invention also includethose in which the buffer is a histidine buffer at a concentration of5-20 mM. In one such embodiment (a) the aflibercept concentration is20-80 mg/mL; (b) the histidine buffer concentration is about 10 mM; (c)the non-ionic surfactant is polysorbate 20 at a concentration of about0.03% (w/v); (d) the tonicifying agent is:

(i) sucrose or trehalose at a concentration of about 9% (w/v), or(ii) proline at a concentration of about 3% (w/v); (e) the concentrationof chloride anion is less than about 10 mM, or more preferably, lessthan about 5 mM; and the pH of the formulation is about pH 5.5 to aboutpH 6.5, or in some embodiments about pH 6.0 to about pH 6.5. In somepreferred embodiments of this formulation, the tonicifying agent issucrose or trehalose at a concentration of about 9% (w/v), with theaflibercept concentration at about 30 mg/mL to about 50 mg/mL; forexample, a concentration of about 40 mg/mL. In other preferredembodiments the tonicifying agent is proline at a concentration of about3% (w/v), with the aflibercept concentration at about 30 mg/mL to about50 mg/mL; for example, a concentration of about 40 mg/mL.

Still other exemplary ophthalmic formulations of the present inventioninclude those in which the buffer is an acetate buffer. In one suchembodiment (a) the aflibercept concentration is 20-80 mg/mL; (b) theacetate buffer is about 10 mM; (c) the non-ionic surfactant ispolysorbate 20, or polysorbate 80, or a poloxamer, e.g., Poloxamer 188,at a concentration of about 0.01% (w/v), about 0.03% (w/v), about 0.1(w/v), or about 1% (w/v); (d) the tonicifying agent is:

(i) sucrose or trehalose at a concentration of about 9% (w/v), or(ii) proline at a concentration of about 3% (w/v); (e) the concentrationof chloride anion is less than about 1 mM; and the pH of the formulationis about pH 5.0 to about pH 5.5. In some preferred embodiments of thisformulation, the tonicifying agent is sucrose or trehalose at aconcentration of about 9% (w/v), with the aflibercept concentration atabout 30 mg/mL to about 50 mg/mL; for example, a concentration of about40 mg/mL. In other preferred embodiments the tonicifying agent isproline at a concentration of about 3% (w/v), with the afliberceptconcentration at about 30 mg/mL to about 50 mg/mL; for example, aconcentration of about 40 mg/mL.

By way of further illustration, the following numbered embodiments areencompassed by the present invention:

Embodiment 1

An ophthalmic formulation, comprising:

(a) aflibercept in a concentration of 5-100 mg/mL;(b) a buffer at 5-50 mM concentration;(c) a non-ionic surfactant;(d) a tonicifying agent selected from the group consisting of a polyoland an amino acid,

wherein the formulation has a final osmolality of about 300 mOsm/kg, and

(e) wherein the concentration of chloride anion is less than about 10mM; andwherein the pH of the formulation is about pH 5.0 to about pH 6.5.

Embodiment 2

The ophthalmic formulation of Embodiment 1, wherein the concentration ofchloride anion is less than about 5 mM.

Embodiment 3

The ophthalmic formulation of Embodiments 1-2, wherein the concentrationof chloride anion is less than about 1 mM.

Embodiment 4

The ophthalmic formulation of Embodiments 1-3, wherein the buffer is aphosphate buffer.

Embodiment 5

The ophthalmic formulation of Embodiments 1-3, wherein the buffer is ahistidine buffer at a concentration of 5-20 mM.

Embodiment 6

The ophthalmic formulation of Embodiments 1-3, wherein the buffer is anacetate buffer.

Embodiment 7

The ophthalmic formulation of Embodiments 1-3, wherein the buffer isselected from phosphate, histidine, acetate, succinate, citrate,glutamate, and lactate, or is a combination of two or more of these.

Embodiment 8

The ophthalmic formulation of Embodiments 1-7, wherein the bufferconcentration is 5-20 mM.

Embodiment 9

The ophthalmic formulation of Embodiments 1-8, wherein the non-ionicsurfactant is selected from the group consisting of a polysorbate (e.g.,polysorbate 20 or polysorbate 80), a polyethylene glycol dodecyl ether(i.e., Brij®35), a poloxamer (e.g., Poloxamer 188),4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (i.e., Triton™X-100), an alkylsaccharide and an alkylglycoside.

Embodiment 10

The ophthalmic formulation of Embodiments 1-9, wherein the non-ionicsurfactant is Poloxamer 188.

Embodiment 11

The ophthalmic formulation of Embodiments 1-10, wherein the tonicifyingagent is a polyol selected from sucrose, trehalose, sorbitol, mannitol,and glycerol.

Embodiment 12

The ophthalmic formulation of Embodiments 1-11, wherein the tonicifyingagent is sucrose.

Embodiment 13

The ophthalmic formulation of Embodiments 1-11, wherein the tonicifyingagent is trehalose.

Embodiment 14

The ophthalmic formulation of Embodiment 11, further comprising anadditional amino acid stabilizing agent.

Embodiment 15

The ophthalmic formulation of Embodiment 14, wherein the additionalamino acid stabilizing agent is selected from the group consisting ofproline, arginine, methionine, glycine, and lysine.

Embodiment 16

The ophthalmic formulation of Embodiments 1-15, wherein the tonicifyingagent is an amino acid selected from proline, arginine, aspartate,glutamate, glycine, histidine, isoleucine, and lysine.

Embodiment 17

The ophthalmic formulation of Embodiments 1-16, wherein the tonicifyingagent is proline.

Embodiment 18

The ophthalmic formulation of Embodiment 4, wherein:

(a) the aflibercept concentration is 20-80 mg/mL;(b) the phosphate buffer concentration is about 10 mM,(c) the non-ionic surfactant is a polysorbate or a poloxamer,(d) the tonicifying agent is (i) sucrose or trehalose at a concentrationof about 9% (w/v) or (ii) proline at a concentration of about 3% (w/v);(e) the concentration of chloride anion is less than about 1 mM;and the pH of the ophthalmic formulation is about pH 6.0 to about pH6.5.

Embodiment 19

The ophthalmic formulation of Embodiment 18, wherein the tonicifyingagent is sucrose or trehalose at a concentration of about 9% (w/v).

Embodiment 20

The ophthalmic formulation of Embodiment 18, wherein the tonicifyingagent is proline at a concentration of about 3% (w/v).

Embodiment 21

The ophthalmic formulation of Embodiment 5, wherein:

(a) the aflibercept concentration is 20-80 mg/mL;(b) the histidine buffer is about 10 mM;(c) the non-ionic surfactant is a polysorbate or a poloxamer;(d) the tonicifying agent is (i) trehalose at a concentration of about9% (w/v) or (ii) proline at a concentration of about 3% (w/v);and the pH of the ophthalmic formulation is about pH 5.5 to about pH6.5.

Embodiment 22

The ophthalmic formulation of Embodiment 21, wherein the tonicifyingagent is trehalose at a concentration of about 9% (w/v).

Embodiment 23

The ophthalmic formulation of Embodiment 21, wherein the tonicifyingagent is proline at a concentration of about 3% (w/v).

Embodiment 24

The ophthalmic formulation of Embodiment 6, wherein:

(a) the aflibercept concentration is 20-80 mg/mL;(b) the acetate buffer is about 10 mM;(c) the non-ionic surfactant is a polysorbate or a poloxamer;(d) the tonicifying agent is (i) sucrose or trehalose at a concentrationof about 9% (w/v) or (ii) proline at a concentration of about 3% (w/v);(e) the concentration of chloride anion is less than about 1 mM;and the pH of the ophthalmic formulation is about pH 5.0 to about pH5.5.

Embodiment 25

Use of any of the formulations of Embodiments 1-24 for the treatment ofan eye disorder or disease.

Embodiment 26

The use of Embodiment 25, wherein the eye disorder or disease isselected from macular edema following Retinal Vein Occlusion (RVO),Central Retinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion(BRVO), Neovascular (Wet) Age-Related Macular Degeneration (AMD),Impaired vision due to Myopic Choroidal Neovascularisation, DiabeticMacular Edema (DME), Diabetic Retinopathy (DR) in patients with DME, andneovascular Age-Related Macular Degeneration (AMD).

Embodiment 27

The use of Embodiments 25-26, wherein the ophthalmic formulation isadministered to a patient with the eye disorder or disease byintravitreal injection.

The following working examples are illustrative and not to be construedin any way as limiting the scope of the invention.

EXAMPLES Example 1. Stability Studies

Materials.

The VEGF-specific aflibercept fusion protein antagonist was producedusing industry standard recombinant expression technology andpurification processes. The purified drug substance was buffer exchangedagainst the specified formulation buffers using a bench scale tangentialflow filtration system by Millipore Corporation. Protein concentrationswere adjusted to the final target concentration by diluting withformulation buffer. The water used in making all formulations waspurified by a Milli-Q® (Millipore Corporation) water purificationsystem, which includes an ion exchange cartridge. The purity of thewater was monitored by measuring the conductivity, with a value greaterthan 18.2 MΩ cm−1 (@ 25 Å° C.) being acceptable. All excipients, buffersand other ingredients used for the preparation of formulation bufferswere USP grade or equivalent.

Methods.

Titrations:

Acid and base conjugates, prepared at equal molarity, were blendedtogether at the appropriate molar ratios to achieve the desiredformulation pH for the histidine and phosphate buffer systems. Theacetate formulations were prepared using a glacial acetic acid additionto Milli-Q-purified water followed by a sodium hydroxide titration toreach the desired final pH.

Appearance:

A qualitative visual appearance test was performed to assess the drugproduct for protein particles or environment contaminants An aliquot(1.1 mL) of protein sample was placed into a pre-sterilized Type 1 glassvial and capped with a 13 mm closure. Under ambient light conditions,the sample was gently swirled and visually inspected for 5 seconds todetect visible particles. For all samples tested, the number anddescription of any detected particles were recorded.

Color.

A qualitative visual color assessment was performed to monitor drugproduct color during product stability. Commercially available EP 2.2.2brown-yellow color standards (BY1-BY7) from Ricca Chemicals werealiquoted into pre-sterilized Type 1 glass vials. An aliquot (1.1 mL) ofdrug product was placed into a vial and compared against an equal volumeof each brown-yellow color standard to determine the level ofcoloration. All samples were inspected in front of a white backgroundand the level of coloration was recorded for each sample.

Reduced Volume Light Obscuration Sub-Visible Particle Analysis.

A HIAC counter equipped with an HRLD-150 sensor (Beckman Corporation)was used to measure subvisible particles by light obscuration. Thesensor was calibrated using polystyrene beads ranging from 1-100 μm.Prior to analysis, MilliQ water was flushed through the system until aclean baseline was achieved and a standard solution of 15 μm polystyrenebeads was used to confirm system suitability. Replicate samples werepooled to a final volume of 1.1 mL in a glass vial and vacuum degassedfor 2 hours at 75 Torr. For each sample, the instrument was set to drawfive aliquots of 0.2 mL and measure particles greater than 10 μm and 25μm. The average of the last three measurement were reported.

Size Exclusion Chromatography.

Size exclusion high performance liquid chromatography (SE-HPLC) analysiswas performed using a Waters XBridge Protein BEH SEC 200A column.Separation was achieved under native conditions using a phosphate,sodium chloride running buffer. Peak elution was detected by UVabsorbance, and the integrated purity results were reported as relativepeak area percentages of the high molecular weight (HMW) component, maincomponent (monomer), and low molecular weight (LMW) component, relativeto total corrected area.

CZE Chloride Ions Analysis.

Chloride ion analysis was performed using a Microsolv TechnologiesCElixirOA pH 5.4 kit run on the Beckman Pa. 800 capillaryelectrophoresis instrument. Samples and standards were preparedaccording to the manufacturer's instructions. Injection was by pressureat 1 psi for 10 seconds onto a 50.2 cm effective length bare fusedcapillary. Samples were monitored by a photo diode array detector (PDA).Analysis of a standard curve was used to quantify the concentration ofchloride anions in each sample.

Reduced Capillary Electrophoresis—Sodium Dodecyl Sulfate (rCE SDS).

Protein samples were denatured by heating at 70° C. for 10 minutes inSDS and reduced with β-mercapto-ethanol. Samples were electrokineticallyinjected into a 30.2 cm bare fused silica capillary filled with SDS gelbuffer. An electrical voltage of −15 kV was applied across thecapillary, which separates species by their difference in size. Proteinswere detected using a photodiode array detector. The purity wasevaluated by determining the percent peak area of each component.

Non-Reduced Capillary Electrophoresis—Sodium Dodecyl Sulfate (nrCE SDS).

Samples were denatured by heating at 60° C. for 5 minutes in thepresence of SDS and N-ethylmaleimide (NEM) at low pH. The resultingnegatively charged SDS-protein complex was electrokinetically injectedinto a 30.2 cm bare-fused silica capillary filled with SDS gel buffer.An electrical voltage of −15 kV was applied across the capillary, whichseparates species by their difference in size. Protein species weredetected by a photodiode array (PDA) detector as they passed through thedetection window. Purity was evaluated by determining the percent peakarea of each component.

Capillary Isoelectric Focusing.

Capillary Isoelectric focusing (cIEF) was employed to separate proteinsbased on differences in isoelectric point (pI). The neutral coatedcapillary was filled with an ampholytic solution and an electric voltagewas applied which focuses the ampholytic species into a pH gradient. Theprotein species were focused into the portion of the pH gradient wherethe pH is equal to their isoelectric point. Proteins were chemicallymobilized and detected by UV absorbance (280 nm) as they passed througha detection window. Purity was evaluated by determining the percent peakarea of each component.

Potency Evaluation.

Potency of aflibercept was assessed in a cell-based VEGF-A165 dependentproliferation assay. Human umbilical vein endothelial cells were platedin 96-well plates in the absence of growth factors with varyingconcentrations of drug product and 100 ng/mL of VEGF-A165. Assay plateswere incubated for approximately 3 days at 37° C., 5% CO before theaddition of a fluorescent viability reagent. Dose response curves weregenerated by graphing drug product concentration versus fluorescence andthen fitting with a 4-parameter fit equation. Relative potency wasmeasured by evaluating a shift along the x-axis between the test sampleand reference standard.

Osmolality Measurements.

The osmolality of the samples was measured using an AdvancedInstruments, Inc. freezing point osmometer. Prior to sample analysis,the instrument calibration was checked using a 290 mOsm/kg Clinitrol™290 Reference Solution (Fisher Scientific). A 20-μL aliquot subsamplefrom a sample was transferred to a sample tube and placed into theinstrument for freezing point analysis. Each sample was analyzed intriplicate and the reported results were an average of these values.

Example 2. Stability Studies

Stability analysis of aflibercept was conducted to understand theimportance of chloride ion, pH, buffer, and stabilizer type to thestability of the molecule. The composition of each aflibercept (40mg/mL) formulation and the abbreviation (i.e., formulation number) usedthroughout the results of this Example 2 and FIGS. 1-10 herein areoutlined in Table 1. Amino acids used were in L-isomeric configuration.

TABLE 1 Formulations studied. Aflibercept concentration was 40 mg/mL.Formulation Number Formulation 1 10 mM sodium phosphate, 40 mM sodiumchloride, 5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2 2 10 mMsodium phosphate, 9% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2 310 mM sodium phosphate, 40 mM sodium chloride, 2% (w/v) proline, 0.03%(w/v) polysorbate 20, pH 6.2 4 10 mM sodium phosphate, 3% (w/v) proline,0.03% (w/v) polysorbate 20, pH 6.2 5 10 mM sodium phosphate, 9% (w/v)Trehalose, 0.03% (w/v) polysorbate 20, pH 6.2 6 10 mM histidine, 3%(w/v) proline, 0.03% (w/v) polysorbate 20, pH 6.2 7 10 mM histidine, 9%(w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2 8 10 mM acetate, 9%(w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 5.2

Chloride Ion Detection.

Chloride ions levels were measured by capillary zone electrophoresis.Formulation buffers and the formulated drug products were tested todetermine the level of chloride ions present in each sample. Drugproduct samples formulated with sodium chloride or those formulated withhistidine are expected to have chloride ions present in the buffer anddrug product samples. As shown in Table 2, the formulation buffers withadded sodium chloride had the expected level of chloride ions, whilehistidine formulations without added sodium chloride showed low levelsof chloride ions, a result due to the histidine-mono hydrochloride usedduring the buffer preparation. All formulated drug product samplesdemonstrated slightly higher levels of chloride ions than thecorresponding buffer sample. This result is likely due to residualchloride from the product purification.

TABLE 2 The capillary zone electrophoresis chloride ion results fromformulation buffers versus the formulated drug product afliberceptsamples. Residual chloride ions from the purification process resultedin slightly elevated chloride ion levels in the drug product samplesversus the formulation buffer alone. Formulation Buffer Formulated DrugCl⁻ Ion Concn Product Cl⁻ Ion Formulation (mM) Concn (mM) 1 41.8 42.6 20.0 0.1 3 38.9 45.9 4 0.0 1.4 5 0.0 0.1 6 2.4 4.7 7 2.8 4.6 8 0.0 0.6

Osmolality.

The osmolality of the drug product solutions was measured to ensure thatco-concentration or exclusion of formulation components during the drugproduct preparation process did not result in unacceptable osmolalitylevels for intravitreal injection. The osmolality results for the eightdrug product formulations evaluated in this study showed osmolalitylevels within the acceptable 250-350 mOsm/kg range. A summary of theseresults are shown in Table 3.

TABLE 3 The osmolality of the eight aflibercept formulated drug productsamples were measured by a freezing point osmometer, which indicated allsamples were within the acceptable range. Formulation Osmolality Number(mOsm/kg) 1 258 2 326 3 277 4 302 5 320 6 297 7 330 8 332

Visual Assessment:

Visible Particulates and Color Inspection. The visual assessment of thedrug product sample was conducted to determine the presence or absenceof environmental or product related particles and to evaluate theproduct color over the duration of the stability study. Stabilitysamples stored at 4° C. for 7 weeks, and 30° C. for 4 weeks, wereinspected for visual particles and all samples passed this assessment.

The color inspection of aflibercept drug product stability samples wasconducted by comparing the formulated protein to commercially availablebrown-yellow pharmacopeia color standards (EP 2.2.2). The results fromthis assessment determined that regardless of formulation andtemperature, the drug product color did not change during storage.

Subvisible Particle Testing:

HIAC. Subvisible particle analysis was conducted on the 4° C. stabilitysamples. The 10-μm and 25-μm particle size results are shown in FIG. 1and FIG. 2, respectively. The error bars in FIG. 1 and FIG. 2 representthe standard deviation calculated from three readings. The measuredsubvisible particle levels were well below those mandated by USPmonograph <789> (Particulate Matter In Ophthalmic Solutions). Ten(10)-μm particles were observed for all formulations stored at 4° C.,apart from Formulation 7, a condition that had increasing levels ofsubvisible particles over time. The 25-μm particle levels, shown in FIG.2 were undetectable or at very low levels when stored at 4° C. It shouldbe noted that the error bars for the 25-μm results are larger than thenumber of particles reported and therefore no trends can be concludedfrom these early time point data. SE-HPLC. Size exclusion highperformance liquid chromatography (SE-HPLC) analysis was used toevaluate the level of high molecular weight species (HMW) in thestability samples throughout the duration of the study. SE-HPLC wasperformed at all time points for all temperature conditions. Resultsfrom the 4° C. and 30° C. temperature storage conditions are shown inFIG. 3 and FIG. 4, respectively. After 7 weeks of storage at 4° C.Formulations 1-7 had HMW levels that were indistinguishable from eachother, which are at an equivalent pH, but have differences in buffertype, tonicity modifier and presence of sodium chloride. Surprisingly,Formulation 8, which had a significantly lower pH than the otherformulations, also had dramatically reduced levels of aggregates ascompared to the other formulations. The trends observed at 4° C. werecomparable to those detected after 4 weeks of storage at 30° C.

The SE-HPLC-measured HMW data imply that sodium chloride is notnecessary to stabilize aflibercept during long term storage, for any ofthe studied formulations, which is surprisingly contrary to the teachingof Dix et al. (See, U.S. Pat. No. 8,921,316, Column 6, lines 62-65;stating that “although either NaCl or sucrose can be used as astabilizer, a combination of NaCl and sucrose has been established tostabilize the fusion protein more effectively than either individualstabilizer alone,” which is contrary to the empirical results we reportherein.) Histidine and acetate buffer systems are viable alternatives tophosphate depending on the pH desired. Significantly reduced levels ofaggregate could be obtained using a buffer in the pH range around 5. Inaddition to sucrose, the tonicifying agents proline and trehalose can beused to reach the appropriate osmolality (˜300 mOsm/kg) whilemaintaining or enhancing the aflibercept molecule stability.

Reduced Capillary Electrophoresis Sodium Dodecyl Sulfate (rCE-SDS).

The rCE-SDS method was used to monitor product degradation over time,such as cleavage of the amino acid backbone, with results being reportedas percent purity, i.e. the total percent of heavy chain and lightchain. The 4° C. results are summarized in FIG. 5, and the 30° C. dataare shown in FIG. 6. Regardless of storage temperature, all formulationsstudied, apart from Formulation 7, have similar levels of puritythroughout the tested time points. Numerical differences observedbetween formulations are within the assay variability. Formulation 7showed an increase in product degradation at the 30° C. elevatedtemperature condition, resulting in a decreased percent purity overtime.

Capillary Isoelectric Focusing (cIEF).

Capillary Isoelectric Focusing (cIEF) was used to monitor changes in thedistribution of charge variants over time. To assess the differentformulations, the percent basic species levels and percent acidicspecies levels were plotted against time. An increase or decreases inthese species would indicate a change in protein charge, likelyresulting from a chemical modification of the peptide backbone. As shownin FIG. 7, FIG. 8, FIG. 9, and FIG. 10, there were no observed changesin the distribution of the charge variants over time. These dataindicate no detectable chemical modifications occurred that affected theaflibercept protein's charge.

Example 3. Stability Studies

Further tests of aflibercept stability in various embodiments of theinventive formulation were carried out. Table 4 contains a list ofaflibercept (40 mg/mL) formulations tested and their abbreviations Aminoacids used were in L-isomeric configuration.

TABLE 4 Formulations of aflibercept (40 mg/mL) tested and theirosmolality and associated abbreviations used in Tables 5-15 herein.Osmolality (mOsm/kg ± Abbreviation Formulation SD; n = 3) P62NaSuT 10 mMsodium phosphate, 40 mM 258.7 ± 4.0 sodium chloride, 5% (w/v) sucrose,0.03% (w/v) polysorbate 20, pH 6.2 A52ProPl-1 10 mM acetate, 3% (w/v)proline, 297.7 ± 2.1 1% (w/v) poloxamer 188, pH 5.2 A52ProPl-0.1 10 mMacetate, 3% (w/v) proline, 300.7 ± 2.3 0.1% (w/v) poloxamer 188, pH 5.2A52ProPl-0.01 10 mM acetate, 3% (w/v) proline, 304.7 ± 2.5 0.01% (w/v)poloxamer 188, pH 5.2 A52ProT 10 mM acetate, 3% (w/v) proline, 299.0 ±0.0 0.03% (w/v) polysorbate 80, pH 5.2 P62ProPl-0.1 10 mM sodiumphosphate, 3% (w/v) 307.0 ± 1.7 proline, 0.1% (w/v) poloxamer 188, pH6.2 P62ProT 10 mM sodium phosphate, 3% (w/v) 294.0 ± 0.0 proline, 0.03%(w/v) polysorbate 80, pH 6.2

Materials.

The VEGF-specific aflibercept fusion protein antagonist was producedusing industry standard recombinant expression technology andpurification processes. The purified drug substance was buffer exchangedagainst the specified formulation buffers using either a lab scale or abench scale tangential flow filtration system by Millipore Corporation.Protein concentrations were adjusted to the final target concentrationby diluting with formulation buffer. The water used in making allformulations was purified by a (Millipore Corporation) waterpurification system, which includes an ion exchange cartridge. Thepurity of the water was monitored by measuring the conductivity, with avalue greater than 18.2 MΩ cm−1 (@ 25 Å° C.) being acceptable. Allexcipients, buffers and other ingredients used for the preparation offormulation buffers were USP grade or equivalent.

Methods.

Titrations.

Acid and base conjugates, prepared at equal molarity, were blendedtogether at the appropriate molar ratios to achieve the desiredformulation pH for the histidine and phosphate buffer systems. Theacetate formulations were prepared using the conjugate method or byusing a glacial acetic acid addition to Milli-Q-purified water followedby a sodium hydroxide titration to reach the desired final pH.

Mass Spectrometry Based Multi-Attribute Method (MAM).

Stability samples were denatured with 6.8 M guanidine, reduced with 10mM Dithiothreitol (DTT), and alkylated with 20 mM iodoacetic acid.Excess reagents were removed by size-exclusion based desalting columns.Trypsin was added at a 1:10 enzyme to substrate ratio and samples weredigested for 30 minutes at 37° C. The resulting peptides were separatedby RP-HPLC with a formic acid/acetonitrile (FA/ACN) gradient over a C18column and monitored by mass spectrometry detection using a ThermoFisher Q-Exactive Mass Spectrometer. Identification and quantificationof the individual peptides was performed using Genedata's Expressionistsoftware.

Other analytic methods were performed as described above in Example 1.

Effect of pH on the Stability of Aflibercept.

The effect of pH on the rate of high molecular weight formation asmeasured by SE-HPLC at 4° C. and 30° C. (see, FIG. 11) and sub-visibleparticle formation as measured by HIAC at 4° C. was examined usingaflibercept produced recombinantly in three (3) different lots, atvarying productions scales. Aflibercept produced at the three differentscales showed similar product characteristics including similar levelsof glycosylation across all lots. The aflibercept was then formulatedwith 10 mM acetate, 3% (w/v) proline and 0.1% (w/v) poloxamer 188 tothree (3) different pH values (see, Table 4 for formulationabbreviations). As shown in Table 5 and Table 6 and FIG. 11, the rate ofSE-HPLC-measured HMW formation of aflibercept formulated in 10 mMacetate, 3% (w/v) proline and 0.1% (w/v) poloxamer 188 was not affectedby pH and was consistently lower than the rates of change observed forthe same lots of aflibercept formulated in 10 mM sodium phosphate, 40 mMsodium chloride, 5% (w/v) sucrose and 0.03% (w/v) polysorbate 20, pH6.2. Sub-visible particle formation measured by HIAC was also examinedduring storage at 4° C. and did not significantly change for afliberceptin any formulations during the storage period (Table 7).

TABLE 5 Rate of HMW formation at 4° C. as measured by SE-HPLC; Rate ofChange % High Molecular Weight (% (HMW) formation at 4° C. HMW 0 2 4 6 813 per Lot Formulation weeks weeks weeks weeks weeks weeks week) 2P62NaSuT 0.69 1.54 1.72 1.95 0.090 2 A52ProPl-0.1 0.31 0.42 0.43 0.490.012 (pH 5.0)* 3 P62NaSuT 1.18 1.45 1.56 1.56 1.70 1.96 0.055 3A52ProPl-0.1 0.35 0.44 0.47 0.51 0.54 0.64 0.021 (pH 5.3)* 1 P62NaSuT0.33 0.49 0.60 0.66 0.025 1 A52ProPl-0.1 0.22 0.24 0.32 0.37 0.012 (pH5.6)* *The value in parenthesis refers to the measured pH of thesolution.

TABLE 6 Rate of HMW formation at 30° C. as measured by SE-HPLC; Rate ofChange % High Molecular Weight (% (HMW) formation at 30° C. HMW 0 2 4 68 13 per Lot Formulation weeks weeks weeks weeks weeks weeks week) 2P62NaSuT 0.69 2.44 3.39 3.91 4.63 5.97 0.467 2 A52ProPl-0.1 0.31 0.560.71 0.80 0.95 1.49 0.076 (pH 5.0)* 3 P62NaSuT 1.18 2.24 2.84 3.14 3.644.58 0.246 3 A52ProPl-0.1 0.35 0.69 0.81 0.86 0.96 1.22 0.060 (pH 5.3)*1 P62NaSuT 0.33 0.94 1.31 1.73 2.18 3.39 0.225 1 A52ProPl-0.1 0.22 0.390.51 0.61 0.73 0.95 0.062 (pH 5.6)* *The value in parenthesis refers tothe measured pH of the solution.

TABLE 7 Subvisible particle formation at 4° C. as measured by smallvolume HIAC analysis; Sub-visible particle formation at 4° C. (valuesare cumulative counts ≥ 10 μm) 0 2 4 6 8 13 Lot Formulation weeks weeksweeks weeks weeks weeks 2 P62NaSuT 20.00 13.33 15.00 20.00 2A52ProPl-0.1 20.00 11.67 10.00 3.33 (pH 5.0)* 3 P62NaSuT 16.67 13.3313.33 10.00 8.33 26.67 3 A52ProPl-0.1 3.33 3.33 10.00 8.33 6.67 15.00(pH 5.3)* 1 P62NaSuT 10.00 10.00 13.33 15.00 1 A52ProPl-0.1 48.33 11.678.33 21.67 (pH 5.6)* *The value in parenthesis refers to the measured pHof the solution.

Stability of Aflibercept in Proline and Arginine Formulations ContainingDifferent Non-Ionic Surfactants.

The stability of aflibercept in a formulation containing either prolineor arginine as a tonicifying agent was examined in the presence ofeither of two surfactants, poloxamer 188 or polysorbate 80 (Table 8 andTable 9, respectively; see, Table 4 for formulation abbreviations). HMWspecies formation as measured by SE-HPLC at 30° C. was significantlyreduced when aflibercept was formulated in 10 mM acetate, 3% (w/v)proline, pH 5.2, as compared to 10 mM sodium phosphate, 40 mM sodiumchloride, 5% (w/v) sucrose, pH 6.2, when each was formulated with eitherpolysorbate 80 or Poloxamer 188. Aflibercept in the 10 mM phosphate, 3%(w/v) proline, pH 6.2, showed a similar rate of SE-HPLC-measured HMWformation to that of aflibercept in the 10 mM sodium phosphate, 40 mMsodium chloride, 5% (w/v) sucrose, pH 6.2 formulation (each formulatedwith either polysorbate 80 or Poloxamer 188, as non-ionic surfactant).In contrast, replacement of the proline with arginine as the tonicifyingagent in the acetate formulation at pH 5.2 increased the rate ofSE-HPLC-measured HMW formation by at least 10-fold. However, in the 10mM phosphate buffer, pH 6.2 formulation the arginine showed similarstability to the aflibercept in the 10 mM phosphate, 3% (w/v) proline,pH 6.2 formulation, regardless of which type of non-ionic surfactant waspresent.

TABLE 8 Rate of HMW formation at 30° C. as measured by SE-HPLC.Poloxamer 188 (0.1% (w/v)) was present as the non-ionic surfactant ineach formulation tested. Rate of % High Molecular Weight Change (HMW)formation at 30° C. (% 0 2 4 6 8 10 12 HMW/ Formulation weeks weeksweeks weeks weeks weeks weeks week) A52ProPl-0.1 0.42 0.57 0.71 0.820.91 0.94 0.97 0.05 A52ArgPl-0.1 0.37 1.13 2.32 3.31 4.40 5.68 7.05 0.56P62ProPl-0.1 0.60 1.15 1.42 1.90 2.20 2.38 2.54 0.16 P62ArgPl-0.1 0.480.90 1.29 1.74 1.99 2.38 2.78 0.19 P62NaSuPl-0.1 0.61 1.10 1.36 1.822.11 2.21 2.38 0.15

TABLE 9 Rate of HMW formation at 30° C. as measured by SE-HPLC.Polysorbate 80 (0.03% (w/v)) was present as the non-ionic surfactant ineach formulation tested. Rate of % High Molecular Weight Change (HMW)formation at 30° C. (% Formu- 0 2 4 6 8 10 12 HMW/ lation weeks weeksweeks weeks weeks weeks weeks week) A52ProT 0.44 0.56 0.72 0.84 0.941.04 1.11 0.06 A52ArgT 0.39 1.58 4.24 6.76 9.23 11.24 13.77 1.15 P62ProT0.60 1.11 1.44 1.88 2.10 2.35 2.61 0.16 P62ArgT 0.45 0.90 1.30 1.78 1.992.42 2.82 0.19 P62NaSuT 0.60 1.08 1.40 1.79 1.88 2.25 2.32 0.14

Stability of Aflibercept Following Simulated Shipping and Effect ofSurfactant Concentration.

The stability of aflibercept was characterized in the 10 mM acetate, 3%(w/v) proline, pH 5.2 formulation with various surfactants andconcentrations following simulated shipping. The simulated shippingprotocol was designed to account for multiple modes of transportationthat could potentially damage the product and affect the stabilityprofile. As shown in Table 10, the rate of SE-HPLC-measured HMWformation of aflibercept stored at 4° C. in the 10 mM acetate, 3% (w/v)proline, pH 5.2 formulation was not dependent on the type of surfactantor the concentration of surfactant, and was significantly less than thatobserved for the aflibercept in the 10 mM phosphate, 40 mM sodiumchloride, 5% (w/v) sucrose, pH 6.2 formulation. In line with priorresults, the aflibercept stability in the 10 mM phosphate, 3% (w/v)proline, pH 6.2 was similar to that in the 10 mM phosphate, 40 mM sodiumchloride, 5% (w/v) sucrose, pH 6.2 formulation. Additionally, while therate of HMW formation at 30° C. was faster than that at 4° C., the orderof stability did not change, with the 10 mM acetate, 3% (w/v) proline,pH 5.2 being the most stable (Table 11). Following the simulatedshipping, the sub-visible particle counts measured by HIAC increased toa similar degree across all formulations (Table 12, compare columns for0 weeks control to 0 weeks). The sub-visible particle counts measured byHIAC did not appear to be dependent on formulation. Product potency wasassessed at the 13-week time point for a subset of formulations andstorage temperatures (see, Table 13). No differences in potency weredetected between the 10 mM acetate, 3% (w/v) proline, pH 5.2formulations or the 10 mM phosphate, 3% (w/v) proline, pH 6.2, whencompared with the 10 mM phosphate, 40 mM sodium chloride, 5% (w/v)sucrose, pH 6.2 formulation. These results indicate the various prolineformulations stabilize the protein and maintain functional activity.

Mass Spectrometry Based Multi-Attribute Method (MAM) Analysis.

MAM analysis was conducted to evaluate the change in post translationalmodification levels for attributes such as isomerization of asparticacid residues, deamidation of asparagine, and oxidation of methionine.Samples from three formulations were evaluated after 3 months of storageat 4° C., 30° C. and 40° C. compared to a −70° C. control sample. Inthis analysis, the 10 mM acetate, 3% (w/v) proline, 0.1% (w/v) poloxamer188, pH 5.2 and the 10 mM acetate, 3% (w/v) proline, 0.03% (w/v)polysorbate 80, pH 5.2 were compared with the aflibercept commercialformulation 10 mM sodium phosphate, 40 mM sodium chloride, 5% (w/v)sucrose, 0.03% (w/v) polysorbate 20, pH 6.2.

Equivalent levels of methionine oxidation were observed between the −70°C. control and the 4° C. stability samples for the three formulations.Higher levels of oxidation were observed in all three formulations after30° C. and 40° C. storage, with the lowest level of oxidation reportedfor the 10 mM acetate, 3% (w/v) proline, 0.1% (w/v) poloxamer 188, pH5.2. The two formulations with higher levels of methionine oxidation,contained polysorbate, an excipient known to promote oxidation ofproteins (Kerwin, Polysorbates 20 and 80 Used in the Formulation ofProtein Biotherapeutics: Structure and Degradation Pathways, Journal ofPharmaceutical Sciences, (2008) 97, 8:2924-2934).

The deamidation levels were assessed for the three formulations after 3months of 4° C., 30° C. and 40° C. storage. No detectable differenceswere observed between the 4° C. samples however samples stored at theelevated temperatures, had significant increases in deamidation levels.For example, the 40° C. aflibercept formulation (10 mM sodium phosphate,40 mM sodium chloride, 5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH6.2.) had 7.6% deamidation on position N84, whereas the 10 mM acetate,3% (w/v) proline, 0.1% (w/v) poloxamer 188, pH 5.2 and the 10 mMacetate, 3% (w/v) proline, 0.03% (w/v) polysorbate 80, pH 5.2formulations had 2.8% and 3.0% N84 deamidation, respectively. Across allfive detected deamidation sites, the 10 mM acetate, 3% (w/v) proline, pH5.2 formulations had significantly lower deamidation after elevatedtemperatures storage than the phosphate formulation at pH 6.2.

The level of isomerized aspartic acid residues was also evaluated duringthe MAM analysis. Six (6) aspartic residues were susceptible toisomerization. Regardless of temperature or formulation, the measureddifferences in % isomerization levels were within the error of themeasurement technique, therefore conclusions could not be drawn fromthese data.

In summary, the MAM data indicate the 10 mM acetate, 3% (w/v) proline,0.1% (w/v) poloxamer 188, pH 5.2 formulation had a lower or equivalentrate of post-translational modification formation than the commercialaflibercept formulation 10 mM sodium phosphate, 40 mM sodium chloride,5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2.

TABLE 10 Rate of HMW formation at 4° C. as measured by SE-HPLC; % HighMolecular Weight (HMW) formation at 4° C. Rate of 0 Change weeks 0 2 4 68 13 (% HMW/ Formulation control* weeks weeks weeks weeks weeks weeksweek) P62NaSuT 1.18 1.27 1.48 1.48 1.48 1.81 1.96 0.052 A52ProPl-1 0.370.37 0.47 0.48 0.48 0.61 0.67 0.022 A52ProPl-0.1 0.35 0.37 0.44 0.430.46 0.56 0.63 0.020 A52ProPl-0.01 0.33 0.35 0.43 0.43 0.47 0.56 0.630.021 A52ProT 0.35 0.39 0.44 0.44 0.46 0.55 0.60 0.017 P62ProPl-0.1 1.061.18 1.30 1.34 1.36 1.64 1.76 0.046 P62ProT 1.09 1.11 1.27 1.32 1.331.56 1.74 0.047 *Control refers to material that was held at 4° C.during the shipping studies without undergoing the simulated transporttreatment. (See, Table 4 for formulation abbreviations.)

TABLE 11 Rate of HMW formation at 30° C. as measured by SE-HPLC; % HighMolecular Weight (HMW) formation at 30° C. Rate of 0 Change weeks 0 2 46 8 13 (% HMW/ Formulation control* weeks weeks weeks weeks weeks weeksweek) P62NaSuT 1.18 1.27 2.22 2.69 3.01 3.63 4.45 0.234 A52ProPl-1 0.370.37 0.70 0.78 0.86 1.02 1.27 0.064 A52ProPl-0.1 0.35 0.37 0.68 0.770.82 0.97 1.22 0.059 A52ProPl-0.01 0.33 0.35 0.68 0.78 0.84 0.99 1.220.061 A52ProT 0.35 0.39 0.67 0.79 0.84 1.03 1.2 0.059 P62ProPl-0.1 1.061.18 1.98 2.46 2.79 3.31 4.14 0.220 P62ProT 1.09 1.11 1.99 2.54 2.873.35 4.15 0.224 *Control refers to material that was held at 4° C.during the shipping studies without undergoing the simulated transporttreatment. (See, Table 4 for formulation abbreviations.)

TABLE 12 Rate of sub-visible particle formation at 4° C. as measured byHIAC; Sub-visible particle formation at 4° C. (values are cumulativecounts ≥ 10 μm) 0 weeks 0 2 4 6 8 13 control* weeks weeks weeks weeksweeks weeks P62NaSuT 16.67 20.00 11.67 23.33 25.00 20.00 15.00A52ProPl-1 3.33 30.00 23.33 20.00 48.33 18.33 21.67 A52ProPl-0.1 3.3340.00 15.00 16.67 28.33 8.33 10.00 A52ProPl-0.01 3.33 25.00 20.00 11.6745.00 8.33 13.33 A52ProT 5.00 8.33 16.67 15.00 13.33 3.33 11.67P62ProPl-0.1 8.33 28.33 40.00 50.00 31.67 30.00 10.00 P62ProT 13.3318.33 40.00 11.67 13.33 11.67 5.00 *Control refers to material that washeld at 4° C. during the shipping studies without undergoing thesimulated transport treatment. (See, Table 4 for formulationabbreviations).

TABLE 13 Relative Potency; % Relative Potency-13-week Time Point −70° C.SD 4° C. SD 30° C. SD P62NaSuT 98.8 8.4 101.2 2.5 NT* A52ProPl-0.1 95.54.2 101.2 3.2 95.1 2.5 A52ProPl-0.01 NT* 115.8 13.7 NT* A52ProT NT*105.0 5.9 94.0 10.1 P62ProPl-0.1 93.8 14.1 94.6 6.2 NT* P62ProT NT* 93.56.9 NT* *NT-All temperatures were not tested by the potency assay;otherwise n = 3.

Comparison of Recombinantly Produced Aflibercept in an InventiveAcetate-Buffered Formulation Compared to Commercially Obtained Eylea®.

The stability of recombinantly produced aflibercept at 30° C. in the 10mM acetate, 3% (w/v) proline, pH 5.2, 0.1% (w/v) poloxamer formulationwas compared to that of Eylea® (aflibercept; Regeneron Pharmaceuticals,Inc., Tarrytown, N.Y.; see, Table 14). Unless particularly noted hereinas being commercially obtained, aflibercept used in all experimentsdescribed herein was produced recombinantly for these studies by JustBiotherapeutics, Inc. (Seattle, Wash.) and was formulated in theformulations described in Table 1 and Table 4 herein. Eylea® drugproduct was purchased from the European market and placed on stabilityin its own container. Samples were removed from the vial at theindicated time points and analyzed by SE-HPLC to characterize the amountof HMW species present. The data in Table 14 demonstrate thataflibercept produced by Just Biotherapeutics and formulated in theindicated formulation had lower % HMW and a lower rate of HMW speciesformation than aflibercept in the Eylea® drug product.

TABLE 14 Comparison of the rate of HMW formation (SE-HPLC) at 30° C. foran embodiment of the inventive formulation and a commercially obtainedaflibercept formulation (Eylea ®). (See, Table 4 for formulationabbreviations.) % High Molecular Weight (HMW) formation at 30° C. Rateof (after indicated number of weeks) Change (% Formulation 0 2 4 6 8 1317 21 HMW/week) A52ProPl-0.1 0.22 0.39 0.51 0.61 0.73 0.95 — — 0.055Eylea ® 1.03 1.74 2.03 2.41 2.83 2.79 3.84 4.85 0.172

Effect of Salt on the Stability of Aflibercept Formulation.

The effect of salt on the stability of aflibercept was investigated inthe 10 mM acetate, 3% (w/v) proline, pH 5.2 formulation during storageat 30° C. As shown in Table 15, the addition of 100 mM sodium chloridesalt to the formulation increased the rate of SE-HPLC-measured HMWspecies formation by 3.8-fold.

TABLE 15 Comparison of the rate of HMW formation (SE-HPLC) at 30° C. foran embodiment of the inventive formulation with or without 100 mM sodiumchloride salt Rate of Change % High Molecular Weight (% (HMW) formationat 30° C. HMW/ Formulation 0 weeks 2 weeks 4 weeks 6 weeks 8 weeks Week)A52ProPl-0.1 0.37 0.63 0.83 0.90 0.99 0.076 (Control) A52ProPl-0.1, 0.381.28 1.93 2.36 2.87 0.303 100 mM sodium chloride

Stability Comparison of Commercially Obtained Aflibercept withRecombinant aflibercept used in these experiments.

Aflibercept, purchased commercially as Zaltrap®, (ziv-aflibercept;Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.) was reprocessed toremove the commercial formulation components (other thanziv-aflibercept), and reformulated into the 10 mM phosphate, 40 mMsodium chloride, 5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2formulation (P62NaSuT). The commercially obtained reprocessedaflibercept and recombinant aflibercept, produced for these studies byJust Biotherapeutics, Inc. and formulated in 10 mM phosphate, 40 mMsodium chloride, 5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2,were incubated at 30° C. and the HMW formation was monitored. As shownin Table 16, the rate of HMW formation of the aflibercept purified fromcommercially obtained Zaltrap® was slightly faster than that observedfor aflibercept produced by Just Biotherapeutics. These resultsdemonstrate that there was not a substantial difference in the inherentaggregation rate of aflibercept fusion proteins obtained commerciallycompared to aflibercept protein used in the experiments describedherein.

TABLE 16 HMW formation (SE-HPLC) at 30° C. of commercially obtainedziv-aflibercept (Zaltrap ®) compared with recombinantly producedaflibercept (by Just Biotherapeutics, Inc.). Rate of Change % HighMolecular Weight (% Formulation (HMW) formation at 30° C. HMW/ inP62NaSuT 0 weeks 2 weeks 4 weeks 6 weeks 8 weeks Week) Aflibercept: 2.082.34 2.94 3.84 4.26 0.29 commercially obtained and reformulatedAflibercept: 0.60 1.24 1.61 — 2.33 0.21 recombinantly produced

Example 4. Tolerability Study of Multiple Placebo Formulations byIntravitreal Administration in Rabbits

To determine the tolerability of some embodiments of the inventiveformulations intended for use with an aflibercept drug product,intravitreal injections of placebo formulations were administered as asingle dose to rabbits, at Charles River Laboratories, Inc., 640 N.Elizabeth Street, Spencerville, Ohio 45887, United States of America, asshown in Table 17.

TABLE 17 Placebo formulations tested as a single dose in male rabbits.Dose Number of Group Volume animals No. Test Material (mL) (Males) 1Placebo 1 (10 mM Phosphate, 0.05 3 40 mM NaCl, 5% (w/v) Sucrose, 0.03%(w/v) polysorbate 20, pH 6.2) 2 Placebo 2 (10 mM Acetate, 3% 0.05 3(w/v) Proline, 0.1% (w/v) poloxamer 188, pH 5.2) 3 Placebo 3 (10 mMAcetate, 3% 0.05 3 (w/v) Proline, 0.03% (w/v) polysorbate 80, pH 5.2) 4Placebo 4 (10 mM Phosphate, 0.05 3 3% (w/v) Proline, 0.1% (w/v)poloxamer 188, pH 6.2) 5 Placebo 5 (10 mM Phosphate, 0.05 3 3% (w/v)Proline, 0.03% (w/v) polysorbate 80, pH 6.2)

The following parameters and end points were evaluated as per the studydesign: clinical signs, body weights, body weight gains, foodconsumption, and ophthalmology.

Among the subject rabbits in the study, there were no early deaths, notreatment-related clinical signs, and no effects on body weight, bodyweight gain, food consumption, nor any ophthalmic findings. Inconclusion, administration of all placebo formulations by intravitrealinjection was well tolerated in rabbits.

We claim:
 1. An ophthalmic formulation, comprising: (a) aflibercept in aconcentration of 5-100 mg/mL; (b) a buffer at 5-50 mM concentration; (c)a non-ionic surfactant; (d) a tonicifying agent selected from the groupconsisting of a polyol and an amino acid, wherein the formulation has afinal osmolality of about 300 mOsm/kg, and (e) wherein the concentrationof chloride anion is less than about 10 mM; and wherein the pH of theformulation is about pH 5.0 to about pH 6.5.
 2. The ophthalmicformulation of claim 1, wherein the concentration of chloride anion isless than about 5 mM.
 3. The ophthalmic formulation of claim 1, whereinthe concentration of chloride anion is less than about 1 mM.
 4. Theophthalmic formulation of claim 1, wherein the buffer is a phosphatebuffer.
 5. The ophthalmic formulation of claim 1, wherein the buffer isa histidine buffer at a concentration of 5-20 mM.
 6. The ophthalmicformulation of claim 1, wherein the buffer is an acetate buffer.
 7. Theophthalmic formulation of claim 1, wherein the buffer is selected fromphosphate, histidine, acetate, succinate, citrate, glutamate, andlactate, or is a combination of two or more of these.
 8. The ophthalmicformulation of claim 1, wherein the buffer concentration is 5-20 mM. 9.The ophthalmic formulation of claim 1, wherein the non-ionic surfactantis selected from the group consisting of a polysorbate, a polyethyleneglycol dodecyl ether, a poloxamer,4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, analkylsaccharide and an alkylglycoside.
 10. The ophthalmic formulation ofclaim 9, wherein the non-ionic surfactant is Poloxamer
 188. 11. Theophthalmic formulation of claim 1, wherein the tonicifying agent is apolyol selected from sucrose, trehalose, sorbitol, mannitol, andglycerol.
 12. The ophthalmic formulation of claim 1, wherein thetonicifying agent is sucrose.
 13. The ophthalmic formulation of claim 1,wherein the tonicifying agent is trehalose.
 14. The ophthalmicformulation of claim 11, further comprising an additional amino acidstabilizing agent.
 15. The ophthalmic formulation of claim 14, whereinthe additional amino acid stabilizing agent is selected from the groupconsisting of proline, arginine, methionine, glycine, and lysine. 16.The ophthalmic formulation of claim 1, wherein the tonicifying agent isan amino acid selected from proline, arginine, aspartate, glutamate,glycine, histidine, isoleucine, and lysine.
 17. The ophthalmicformulation of claim 16, wherein the tonicifying agent is proline. 18.The ophthalmic formulation of claim 4, wherein: (a) the afliberceptconcentration is 20-80 mg/mL; (b) the phosphate buffer concentration isabout 10 mM, (c) the non-ionic surfactant is a polysorbate or apoloxamer, (d) the tonicifying agent is (i) sucrose or trehalose at aconcentration of about 9% (w/v) or (ii) proline at a concentration ofabout 3% (w/v); (e) the concentration of chloride anion is less thanabout 1 mM; and the pH of the formulation is about pH 6.0 to about pH6.5.
 19. The ophthalmic formulation of claim 18, wherein the tonicifyingagent is sucrose or trehalose at a concentration of about 9% (w/v). 20.The ophthalmic formulation of claim 18, wherein the tonicifying agent isproline at a concentration of about 3% (w/v).
 21. The ophthalmicformulation of claim 5, wherein: (a) the aflibercept concentration is20-80 mg/mL; (b) the histidine buffer is about 10 mM; (c) the non-ionicsurfactant is a polysorbate or a poloxamer; (d) the tonicifying agent is(i) trehalose at a concentration of about 9% (w/v) or (ii) proline at aconcentration of about 3% (w/v); and the pH of the formulation is aboutpH 5.5 to about pH 6.5.
 22. The ophthalmic formulation of claim 21,wherein the tonicifying agent is trehalose at a concentration of about9% (w/v).
 23. The ophthalmic formulation of claim 21, wherein thetonicifying agent is proline at a concentration of about 3% (w/v). 24.The ophthalmic formulation of claim 6, wherein: (a) the afliberceptconcentration is 20-80 mg/mL; (b) the acetate buffer is about 10 mM; (c)the non-ionic surfactant is a polysorbate or a poloxamer; (d) thetonicifying agent is (i) sucrose or trehalose at a concentration ofabout 9% (w/v) or (ii) proline at a concentration of about 3% (w/v); (e)the concentration of chloride anion is less than about 1 mM; and the pHof the formulation is about pH 5.0 to about pH 5.5.
 25. A method oftreating an eye disorder or disease, comprising administering atherapeutically effective amount of the ophthalmic formulation of claim1, claim 4, claim 5, claim 6, or claim 7 to a patient in need oftreatment.
 26. The method of claim 25, wherein the eye disorder ordisease is selected from the group consisting of macular edema followingRetinal Vein Occlusion (RVO), Central Retinal Vein Occlusion (CRVO),Branch Retinal Vein Occlusion (BRVO), Neovascular (Wet) Age-RelatedMacular Degeneration (AMD), Impaired vision due to Myopic ChoroidalNeovascularisation, Diabetic Macular Edema (DME), Diabetic Retinopathy(DR) in patients with DME, and neovascular Age-Related MacularDegeneration (AMD).
 27. The method of claim 25, wherein administeringthe ophthalmic formulation is by intravitreal injection.